U.S. patent number 9,752,171 [Application Number 14/871,483] was granted by the patent office on 2017-09-05 for pentose fermentation by a recombinant microorganism.
This patent grant is currently assigned to Codexis, Inc.. The grantee listed for this patent is Codexis, Inc.. Invention is credited to Catherine M. Cho, Benjamin N. Mijts, Ezhilkani Subbian, Xiyun Zhang.
United States Patent |
9,752,171 |
Subbian , et al. |
September 5, 2017 |
Pentose fermentation by a recombinant microorganism
Abstract
The present invention provides recombinant nucleic acid
constructs comprising a xylose isomerase polynucleotide, a
recombinant fungal host cell comprising a recombinant xylose
isomerase polynucleotide, and related methods.
Inventors: |
Subbian; Ezhilkani (Mountain
View, CA), Zhang; Xiyun (Fremont, CA), Mijts; Benjamin
N. (San Carlos, CA), Cho; Catherine M. (Daly City,
CA) |
Applicant: |
Name |
City |
State |
Country |
Type |
Codexis, Inc. |
Redwood City |
CA |
US |
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Assignee: |
Codexis, Inc. (Redwood City,
CA)
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Family
ID: |
45004413 |
Appl.
No.: |
14/871,483 |
Filed: |
September 30, 2015 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20160010132 A1 |
Jan 14, 2016 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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14034897 |
Sep 24, 2013 |
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13117278 |
May 27, 2011 |
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61349636 |
May 28, 2010 |
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61444226 |
Feb 18, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12P
7/10 (20130101); C12N 9/90 (20130101); C12P
7/04 (20130101); C12N 9/92 (20130101); C12P
19/02 (20130101); C12N 15/81 (20130101); C12Y
503/01005 (20130101); C12P 19/24 (20130101); Y02E
50/10 (20130101); Y02E 50/17 (20130101); Y02E
50/16 (20130101); C12P 2203/00 (20130101) |
Current International
Class: |
C12N
1/00 (20060101); C12N 9/90 (20060101); C12P
7/04 (20060101); C12P 19/24 (20060101); C12P
7/10 (20060101); C12P 19/02 (20060101); C12N
9/92 (20060101); C12N 15/81 (20060101) |
References Cited
[Referenced By]
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0137280 |
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Apr 1985 |
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EP |
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93/03159 |
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Feb 1993 |
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WO |
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95/22625 |
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Aug 1995 |
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WO |
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96/00787 |
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Jan 1996 |
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WO |
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97/0078 |
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Jan 1997 |
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WO |
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97/35966 |
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Oct 1997 |
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WO |
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98/27230 |
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Jun 1998 |
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WO |
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98/31837 |
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Jul 1998 |
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WO |
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00/04190 |
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Jan 2000 |
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WO |
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00/42651 |
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Jul 2000 |
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WO |
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01/75767 |
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Oct 2001 |
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WO |
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2009/152336 |
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WO |
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2010/000464 |
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WO |
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2010/144103 |
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Dec 2010 |
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WO |
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2011/006136 |
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Jan 2011 |
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WO |
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2012/097091 |
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Jul 2012 |
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WO |
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|
Primary Examiner: Hutson; Richard
Attorney, Agent or Firm: Codexis, Inc.
Parent Case Text
The present application is a Divisional of co-pending U.S. patent
application Ser. No. 14/034,897, filed Sep. 24, 2013, which is a
Divisional of Ser. No. 13/117,278, filed May 27, 2011, which claims
priority to U.S. Prov. Appln. Ser. No. 61/349,636, filed on May 28,
2010 and U.S. Prov. Appln. Ser. No. 61/444,226, filed on Feb. 18,
2011, all of which are incorporated by reference in their
entireties.
Claims
We claim:
1. A recombinant fungal host cell comprising a polynucleotide
sequence that encodes a polypeptide that is capable of catalyzing
the isomerization of D-xylose directly to D-xylulose, wherein the
polynucleotide is a recombinant polynucleotide encoding a xylose
isomerase variant.sub.-- polypeptide comprising an amino acid
sequence having at least 90% identity to SEQ ID NO:2, wherein said
polypeptide further comprises the substitutions
T64Q/K233C/F250/E372G, relative to SEQ ID NO:2, wherein the
positions are numbered by correspondence with the amino acid
sequence of SEQ ID NO:2.
2. The recombinant fungal host cell of claim 1, wherein said
isolated xylose isomerase variant is a mature form having xylose
isomerase activity further comprising a substitution at one or more
positions selected from E2, N6, Q13, K16, T18, E29, G62, T67, Q70,
S71, A74, A75, K78, V81, L91, S106, K111, Q116, K127, Q128, A139,
S156, A164, Y182, M199, K201, M206, K211, K223, , T236, K237, T244,
V247, L248, H274, Q277, R281, R284, A325, F328, T329, N330, A339,
G342, G356, F360, I371, D373, R375, K378, V380, D382, S386, T389,
G390, I391, A393, A397, G398, K399, A400, S404, K407, E414, R423,
Q424, M426, V431, N433, V434, L435, and/or F436, wherein the
positions are numbered by correspondence with the amino acid
sequence set forth in SEQ ID NO:2.
3. The recombinant fungal host cell of claim 2, wherein said
variant is a mature form having xylose isomerase activity further
comprising a substitution at one or more positions selected from
E2S, N6G, N6H, Q13K, K16E, T18C, T18K, T18L, T18M, E29N, G62F,
T67S, Q70E, S71L, A74G, A75T, K78R, V81I, L91M, S109D, K111A,
K111L, Q116C, K127I, K127R, Q128A, A139G, S156T, A164V, Y182C,
M199A, M199V, L201H, M206T, K211H, K223T, T236A, T236L, K237A,
T244S, V247A, L248S, H274R, Q277R, R281L, R284H, A325R, A325S,
F328H, T329S, N330G, N330H, N330L, N330W, N330Y, A339R, G342P,
G342V, G356A, F360M, I371G, I371L, I371Q, I371R, I371T, D373G,
R375Q, R375T, R375V, K378A, K378D, V380W, D382G, D382N, S386K,
T389H, G390M, I391A, I391L, A93T, A397L, A397S, G398E, K399E,
K399T, K399V, A400G, S404Y, K407E, K407L, K407R, E414A,R423G,
Q424H, M426R, V431E, N433A, N433H, N433R, V434Q, V434S, L435S,
and/or F436G, wherein the positions are numbered by correspondence
with the amino acid sequence set forth in SEQ ID NO:2.
4. The recombinant fungal host cell of claim 1, wherein the
polynucleotide is integrated into the host cell genome.
5. The recombinant fungal host cell of claim 1, wherein the host
cell is a yeast cell.
6. The recombinant fungal host cell of claim 1, wherein the host
cell has had one or more native genes deleted from its genome.
7. The recombinant fungal host cell of claim 6, wherein the
deletion results in one or more phenotypes including increased
transport of xylose into the host cell, increased xylulose kinase
activity, increased flux through the pentose phosphate pathway,
decreased sensitivity to catabolite repression, increased tolerance
to ethanol, increased tolerance to acetate, increased tolerance to
increased osmolarity, increased tolerance to low pH, and/or reduced
production of by products, wherein comparison is made with respect
to the corresponding host cell without the deletion(s).
8. The recombinant fungal host cell of claim 1, wherein said host
cell is altered to overexpress one or more polynucleotides.
9. The recombinant fungal host cell of claim 8, wherein
overexpression results in one or more phenotypes, including
increased transport of xylose into the host cell, increased
xylulose kinase activity, increased flux through the pentose
phosphate pathway, decreased sensitivity to catabolite repression,
increased tolerance to ethanol, increased tolerance to acetate,
increased tolerance to increased osmolarity, increased tolerance to
low pH, and/or reduced product of by products, wherein comparison
is made to the corresponding unaltered host cell.
10. The recombinant fungal host cell of claim 1, wherein the host
cell is capable of growth in a xylose-based culture medium.
11. The recombinant fungal host cell of claim 1, wherein the host
cell is capable of fermentation in a xylose-based culture
medium.
12. The recombinant fungal host cell of claim 1, wherein the host
cell is capable of faster growth in a xylose-based culture medium
as compared to wild-type Saccharomyces cerevisiae.
13. The recombinant fungal host cell of claim 1, wherein the
xylose-based culture medium is a product from a cellulosic
saccharification process and/or a hemicellulosic feedstock.
Description
REFERENCE TO A "SEQUENCE LISTING," A TABLE, OR A COMPUTER PROGRAM
LISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE
The Sequence Listing written in file CX3-057US_ST25.txt, created on
Jun. 17, 2011, 46,627 bytes, machine format IBM-PC, MS-Windows
operating system, is hereby incorporated by reference.
FIELD OF THE INVENTION
The present invention provides methods and compositions suitable
for use in the isomerization of xylose to xylulose.
BACKGROUND
Ethanol and ethanol fuel blends are widely used in Brazil and in
the United States as a transportation fuel. Combustion of these
fuels is believed to produce fewer of the harmful exhaust emissions
(e.g., hydrocarbons, nitrogen oxide, and volatile organic compounds
(VOCs)) that are generated by the combustion of petroleum.
Bioethanol is a particularly favored form of ethanol because the
plant biomass from which it is produced utilizes sunlight, an
energy source that is renewable. In the United States, ethanol is
used in gasoline blends that are from 5% to 85% ethanol. Blends of
up to 10% ethanol (E10) are approved for use in all gasoline
vehicles in the U.S. and blends of up to 85% ethanol (E85) can be
utilized in specially engineered flexible-fuel vehicles (FFV). The
Brazilian government has mandated the use of ethanol-gasoline
blends as a vehicle fuel, and the mandatory blend has been 25%
ethanol (E25) since 2007.
Bioethanol is currently produced by the fermentation of hexose
sugars that are obtained from carbon feedstocks. Currently, only
the sugar from sugar cane and starch from feedstock such as corn
can be economically converted. There is, however, much interest in
using lignocellulosic feedstocks where the cellulose part of a
plant is broken down to sugars and subsequently converted to
ethanol. Lignocellulosic biomass is made up of cellulose,
hemicelluloses, and lignin. Cellulose and hemicellulose can be
hydrolyzed in a saccharification process to sugars that can be
subsequently converted to ethanol via fermentation. The major
fermentable sugars from lignocelluloses are glucose and xylose. For
economical ethanol yields, a strain that can effectively convert
all the major sugars present in cellulosic feedstock would be
highly desirable.
SUMMARY OF THE INVENTION
The present invention provides methods and compositions suitable
for use in the isomerization of xylose to xylulose.
The present invention provides a recombinant nucleic acid construct
comprising a polynucleotide sequence that encodes a polypeptide
which is capable of catalyzing the isomerization of D-xylose
directly to D-xylulose, wherein the polynucleotide is selected from
a polynucleotide that encodes a polypeptide comprising an amino
acid sequence having at least 70% identity to SEQ ID NO: 2, and/or
a polynucleotide that hybridizes under stringent hybridization
conditions to the complement of a polynucleotide that encodes a
polypeptide having the amino acid sequence of SEQ ID NO: 2.
The present invention further provides a recombinant fungal host
cell transformed with at least one nucleic acid construct of the
present invention.
The present invention further provides a process for producing a
fermentation product, wherein the method comprises: (a) providing a
recombinant host fungal host cell of the present invention; (b)
providing a fermentation medium comprising xylose; and (c)
fermenting the culture medium with the recombinant fungal host cell
under conditions suitable for generating the fermentation
product.
In some embodiments, the polynucleotide sequence encodes a
polypeptide comprising an amino acid sequence at least 70%, at
least 75%, at least 76%, at least 77%, at least 78%, at least 79%,
at least 80%, at least 81%, at least 82%, at least 83%, at least
84%, at least 85%, at least 86%, at least 87%, at least 88%, at
least 89%, at least 90%, at least 91%, at least 92%, at least 93%,
at least 94%, at least 95%, at least 96%, at least 97%, at least
98%, or at least 99% identical to SEQ ID NO:2. In some embodiments,
the polynucleotide sequence encodes a polypeptide comprising the
amino acid sequence of SEQ ID NO:2. In some further embodiments,
the polynucleotide sequence encodes a polypeptide consisting of the
amino acid sequence of SEQ ID NO:2. In some embodiments, the
polynucleotide sequence of the nucleic acid construct is at least
at least 75%, at least 76%, at least 77%, at least 78%, at least
79%, at least 80%, at least 81%, at least 82%, at least 83%, at
least 84%, at least 85%, at least 86%, at least 87%, at least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at
least 98%, or at least 99% identical to SEQ ID NO:1 and/or SEQ ID
NO:3. In some embodiments, the nucleic acid constructs comprise the
nucleotide sequence of SEQ ID NO:1 and/or SEQ ID NO:3.
In some embodiments, the present invention provides at least one
nucleic acid construct comprising a polynucleotide sequence
encoding a polypeptide having an amino acid sequence that comprises
at least one substitution at position 2, 6, 13, 16, 18, 29, 62, 64,
67, 70, 71, 74, 75, 78, 81, 91, 106, 111, 116, 127, 128, 139, 156,
164, 182, 199, 201, 206, 211, 223, 237, 233, 236, 244, 248, 250,
274, 277, 281, 284, 325, 328, 329, 330, 339, 342, 356, 360, 371,
372, 373, 375, 378, 380, 382, 386, 389, 390, 391, 393, 397, 398,
399, 400, 404, 407, 414, 423, 424, 426, 427, 431, 433, 434, 435,
and/or 436, wherein the positions are numbered by correspondence
with the amino acid sequence set forth in SEQ ID NO:2. In some
embodiments, the polynucleotide sequence of the at least one
nucleic acid construct encodes a polypeptide having an amino acid
sequence that comprises at least one substitution selected from E2,
N6, Q13, K16, T18, E29, G62, T64, T67, Q70, S71, A74, A75, K78,
V81, L91, S106, K111, Q116, K127, Q128, A139, S156, A164, Y182,
M199, K201, M206, K211, K223, K233, T236, K237, T244, V247, L248,
F250, H274, Q277, R281, R284, A325, F328, T329, N330, A339, G342,
G356, F360, I371, E372, D373, R375, K378, V380, D382, S386, T389,
G390, I391, A393, A397, G398, K399, A400, S404, K407, E414, R423,
Q424, M426, V431, N433, V434, L435, and/or F436, wherein the
positions are numbered by correspondence with the amino acid
sequence set forth in SEQ ID NO:2. In some further embodiments, the
polynucleotide sequence of the at least one nucleic acid construct
encodes a polypeptide having an amino acid sequence that comprises
at least one substitution selected from E2S, N6G, N6H, Q13K, K16E,
T18C, T18K, T18L, T18M, E29N, G62F, T64Q, T67S, Q70E, S71L, A74G,
A75T, K78R, V81I, L91M, 5109D, K111A, K111L, Q116C, K1271, K127R,
Q128A, A139G, S156T, A164V, Y182C, M199A, M199V, L201H, M206T,
K211H, K223T, K233C, T236A, T236L, K237A, T244S, V247A, L248S,
F250C, F250V, H274R, Q277R, R281L, R284H, A325R, A325S, F328H,
T329S, N330G, N330H, N330L, N330W, N330Y, A339R, G342P, G342V,
G356A, F360M, I371G, I371L, I371Q, I371R, I371T, E372G, E372T,
D373G, R375Q, R375T, R375V, K378A, K378D, V380W, D382G, D382N,
S386K, T389H, G390M, I391A, I391L, A393T, A397L, A397S, G398E,
K399E, K399T, K399V, A400G, 5404Y, K407E, K407L, K407R, E414A,
R423G, Q424H, M426R, V431E, N433A, N433H, N433R, V434Q, V434S,
L435S, and/or F436G, wherein the positions are numbered by
correspondence with the amino acid sequence set forth in SEQ ID
NO:2. In yet some additional embodiments, the polynucleotide
sequence of the at least one nucleic acid construct encodes a
polypeptide having an amino acid sequence that comprises at least
one substitution set selected from N6G/E372G/F436G;
K16E/K111A/E372G; K16E/K111A/E372G/K399T; E29N/E372G;
T64Q/S71L/Q116C/M199A/F360M/E372G/K407R;
T64Q/S71L/Q116C/K233C/F360M/E372G/K407L/Q424H;
T64Q/S71L/M199A/K233C/E372G/1391L; T64Q/S71L/K233C/F360M/E372G;
T64Q/L91M/A139G/A164V/K233C/E372G;
T64Q/Q116C/M199A/F360M/E372G/K407L; T64Q/Q116C/K233C/E372G;
T64Q/M199A/K233C/E372G; T64Q/M199A/K233C/E372G/K407L/Q424H;
T64Q/K233C/F250C/E372G; T64Q/K233C/F360M/E372G/K407L/Q424H;
T64Q/F360M/E372G; T67S/Q70E/A75T/E372G;
T67S/Q70E/S109D/T236A/E372G/S386K;
T67S/Q70E/S109D/T236L/E372G/1391L/G398E/V434S;
T67S/Q70E/S109D/R281L/E372G; T67S/Q70E/S109D/R281L/E372G/S404Y;
T67S/Q70E/S109D/E372G/S386K; T67S/Q70E/S109D/E372G/1391L/S404Y;
T67S/Q70E/S109D/E372G/V431E; T67S/Q70E/S109D/E372G/V434S;
T67S/Q70E/T236A/E372G; T67S/Q70E/T236L/E372G/S386K;
T67S/Q70E/T236L/E372G/V431E; T67S/Q70E/R281L/E372G;
T67S/Q70E/R281L/E372G/S404Y; T67S/Q70E/A325S/E372G;
T67S/Q70E/E372G/S386K; T67S/Q70E/E372G/G390M;
T67S/S109D/R281L/E372G; T67S/S109D/E372G/G398E/V434S;
T67S/R281L/A325R/E372G; Q70E/S109D/T236A/E372G/1391L;
Q70E/S109D/T236A/E372G/V434S; Q70E/S109D/T236L/E372G/S386K/S404Y;
Q70E/S109D/E372G; Q70E/S109D/E372G/G398E; Q70E/S109D/E372G/V431E;
Q70E/T236A/E372G; Q70E/T236A/E372G/G398E;
Q70E/T236A/R281L/A325S/E372G; Q70E/T236L/E372G/G398E;
Q70E/E372G/V434S; Q70E/E372G/G398E/V434S;
S71L/M199A/K233C/E372G/K407L; S71L/E372G; K78R/Y182C/G356A/E372G;
K78R/V247A/L248S/G356A/E372G; K78R/V247A/E372G; K78R/G356A/E372G;
K78R/E372G/K399E/R423G; K78R/D373G; S109D/T236A/R281L/E372G;
S109D/T236L/R281L/A325R/E372G; S109D/R281L/E372G;
Q116C/M199A/K233C/E372G/K407L; Q116C/M199A/F360M/E372G;
K127R/G356A/E372G; K127R/E372G/D373G; Y182C/V247A/G356A;
L201H/E372G; M206T/L248S/H274R/K399E; M206T/L248S/E372G;
K211H/E372G/K407E; K233C/F360M/E372G/V380W/Q424H;
K233C/E372G/V380W; K233C/E372G/K407L;
K223T/K237A/E372G/K399T/K407E; V247A/L248S/G356A/E372G;
R281L/A325S/E372G/A397S; R284H/E372G; T329S/N330H/E372G/R375V;
N330Y/E372G/F436G; G356A/E372G; G356A/E372G/K399E/R423G;
G356A/D373G; F360M/E372G/Q424H; I371G/E372G/N433A; E372G/K378D;
E372G/K378D/K399T/K407E; E372G/1391L/S404Y/V434S; E372G/K399T;
E372G/K399T/K407E; E372G/K407E; E372G/K407R; and/or E372G/L435S,
wherein the positions are numbered by correspondence with the amino
acid sequence set forth in SEQ ID NO:2.
The present invention also provides nucleic acid constructs
comprising polynucleotide sequences that comprise at least one
mutation and/or mutation set selected from
t9c/c12t/c15t/g123a/t132g/a135g/t492a/a606g/c612t;
c15g/t132a/t249a/t252g/c927g/a930g/t1290c;
a48g/c51t/a54g/t57c/t60g/a1209g; a48g/c108a/t882c;
c51a/a54g/g1011a;
a54g/t60a/t168c/t171c/c177t/a180t/c213a/c216t/a219c/g222a/a225c/t891a/c89-
4t/a897c; a54g/g438a/c447t/t450g/c798t/t801c/c804t/c807a;
t102c/c213a/c216t/a219g/g222t/a225c/a813g/a819g/c822t/a825g;
t66a/c138g/t150g/a258g/t261c/t267c/t543g/t546c/c549t;
t66c/c138g/g582a/a987g;
a93t/c96t/t102c/a180g/g768a/t1008c/g1011t/a1014g/t1017g;
a93t/c96t/t102g/a180t/a813g/a819g/a825t; c108g; c108t/c396t/t402c;
t120c/t360a/c993a/c996g/g999a;
g123a/a126g/c129t/t132a/a135c/t1164c/c1167t/t1170g;
g123a/a333g/t403c/c423t/t426c/t429c/c435a/c549g/t552c/t981g/c984t/a987g/t-
990c/a1221g; a126g/t132c/a135c/g438a/c441t/c447t/t450c;
c129t/a135g/c441t; c138a/c147t/t186c/g192t/c858t/t861g/a864g/a987t;
c138a/t150a/c177t/g783a/t1143g/c1146t/c1155a/t1263a/a1269g;
c138a/t150a/g783a/t1143g/c1146t/c1155a/t1263a;
c138a/t150a/g783a/t1143g/c1146t/c1155a/t1263a/a1269g;
c138a/t150a/c307t/g783a/t1143g/c1146t/c1155a/t1263a/a1269g;
t150g/c1146t/t1152c/c1155g; t156c/t165c; t168a/c177t/a420g;
t168c/a180g/a813g/a816c/a819g/c822t/a825g/g1011a/a1014g/t1017a/t1020c;
t168g/a819g/c822t/a825g;
a180t/c291t/c294t/a693g/c696t/a813g/a816t/c822t/a825g; t211a;
c213t/a219g/c339a/a888g/t891g/c894t/a897g/g1011t/t1017a;
c213g/a219g/a225g/c411g/t414c/t417g/g528a/g531a/c534g/a819g/a825g;
g222t/a225g/a453t/t462g/t465g/g528a/g531a/c534g/t537g/c579g/a693g/c696t/a-
774g/c780t/g1134a/g1140a; a228g;
t261a/t309g/t312g/t429c/c432t/c435t/a903g/a906g;
t261a/t543g/t552c/a741c/t870g/t960c/t1026a/a1029t/c1032t/g1035c;
c276t/t279c/c285t/a606g/c828t/a840g/t873a/t882g/c885t;
c288t/c291t/c294t/t300c/a405g/t651c; c307t;
a318g/t558a/t561a/a567g/t570g/t735g/c798g/t801c/c807g/a810g;
g351t/c354t/t360g/c600g; t834c/a840g;
c411t/t414g/t417g/a420g/t429c;
t414g/t417g/a420g/a453c/t459a/t462c/c822t/a825t/t1008c/t1017g/t1020g;
c441t/c447t/a810c/a1095g;
c480t/c522g/t708g/c720t/c762tt960c/t1228c;
a516g/t558g/a564g/c798g/c804t/a810c/a1209t/a1212c;
g528a/t537a/c573t/c579g/g585c/c696a/t705g;
t546c/c549t/c858t/t861g/a864c/t870a; t591g/c600g/a840g;
g654a/t657g; t771c/a774g/c894t/a897g/t1128a/c1131t/t1185c;
a816t/a819g/c822t/g1011t/a1014g; t1065c; a1086g/a1095g; a1125g;
t1137c; and t1263a/t1266g, wherein the nucleotide position is
determined by alignment with SEQ ID NO:1.
The present invention also provides isolated xylose isomerase
variants. In some embodiments, the variants are the mature form
having xylose isomerase activity and comprise at least one
substitution at one or more positions selected from 2, 6, 13, 16,
18, 29, 62, 64, 67, 70, 71, 74, 75, 78, 81, 91, 106, 111, 116, 127,
128, 139, 156, 164, 182, 199, 201, 206, 211, 223, 237, 233, 236,
244, 248, 250, 274, 277, 281, 284, 325, 328, 329, 330, 339, 342,
356, 360, 371, 372, 373, 375, 378, 380, 382, 386, 389, 390, 391,
393, 397, 398, 399, 400, 404, 407, 414, 423, 424, 426, 427, 431,
433, 434, 435, and/or 436, wherein the positions are numbered by
correspondence with the amino acid sequence of SEQ ID NO:2. In some
embodiments, the variant is the mature form, has xylose isomerase
activity, and comprises at least one substitution at one or more
positions selected from E2, N6, Q13, K16, T18, E29, G62, T64, T67,
Q70, S71, A74, A75, K78, V81, L91, S106, K111, Q116, K127, Q128,
A139, S156, A164, Y182, M199, K201, M206, K211, K223, K233, T236,
K237, T244, V247, L248, F250, H274, Q277, R281, R284, A325, F328,
T329, N330, A339, G342, G356, F360, I371, E372, D373, R375, K378,
V380, D382, S386, T389, G390, I391, A393, A397, G398, K399, A400,
S404, K407, E414, R423, Q424, M426, V431, N433, V434, L435, and/or
F436, wherein the positions are numbered by correspondence with the
amino acid sequence set forth in SEQ ID NO:2. In still additional
embodiments, the isolated xylose isomerase variant is a mature form
having xylose isomerase activity and comprising a substitution at
one or more positions selected from E2S, N6G, N6H, Q13K, K16E,
T18C, T18K, T18L, T18M, E29N, G62F, T64Q, T67S, Q70E, S71L, A74G,
A75T, K78R, V81I, L91M, S109D, K111A, K111L, Q116C, K1271, K127R,
Q128A, A139G, S156T, A164V, Y182C, M199A, M199V, L201H, M206T,
K211H, K223T, K233C, T236A, T236L, K237A, T244S, V247A, L248S,
F250C, F250V, H274R, Q277R, R281L, R284H, A325R, A325S, F328H,
T329S, N330G, N330H, N330L, N330W, N330Y, A339R, G342P, G342V,
G356A, F360M, I371G, I371L, I371Q, I371R, I371T, E372G, E372T,
D373G, R375Q, R375T, R375V, K378A, K378D, V380W, D382G, D382N,
S386K, T389H, G390M, I391A, I391L, A393T, A397L, A397S, G398E,
K399E, K399T, K399V, A400G, 5404Y, K407E, K407L, K407R, E414A,
R423G, Q424H, M426R, V431E, N433A, N433H, N433R, V434Q, V434S,
L435S, and/or F436G, wherein the positions are numbered by
correspondence with the amino acid sequence set forth in SEQ ID
NO:2. In some further embodiments, the isolated xylose isomerase
variant is a mature form having xylose isomerase activity and
comprises at least one substitution set selected from
N6G/E372G/F436G; K16E/K111A/E372G; K16E/K111A/E372G/K399T;
E29N/E372G; T64Q/S71L/Q116C/M199A/F360M/E372G/K407R;
T64Q/S71L/Q116C/K233C/F360M/E372G/K407L/Q424H;
T64Q/S71L/M199A/K233C/E372G/1391L; T64Q/S71L/K233C/F360M/E372G;
T64Q/L91M/A139G/A164V/K233C/E372G;
T64Q/Q116C/M199A/F360M/E372G/K407L; T64Q/Q116C/K233C/E372G;
T64Q/M199A/K233C/E372G; T64Q/M199A/K233C/E372G/K407L/Q424H;
T64Q/K233C/F250C/E372G; T64Q/K233C/F360M/E372G/K407L/Q424H;
T64Q/F360M/E372G; T67S/Q70E/A75T/E372G;
T67S/Q70E/S109D/T236A/E372G/S386K;
T67S/Q70E/S109D/T236L/E372G/1391L/G398E/V434S;
T67S/Q70E/S109D/R281L/E372G; T67S/Q70E/S109D/R281L/E372G/S404Y;
T67S/Q70E/S109D/E372G/S386K; T67S/Q70E/S109D/E372G/1391L/S404Y;
T67S/Q70E/S109D/E372G/V431E; T67S/Q70E/S109D/E372G/V434S;
T67S/Q70E/T236A/E372G; T67S/Q70E/T236L/E372G/S386K;
T67S/Q70E/T236L/E372G/V431E; T67S/Q70E/R281L/E372G;
T67S/Q70E/R281L/E372G/S404Y; T67S/Q70E/A325S/E372G;
T67S/Q70E/E372G/S386K; T67S/Q70E/E372G/G390M;
T67S/S109D/R281L/E372G; T67S/S109D/E372G/G398E/V434S;
T67S/R281L/A325R/E372G; Q70E/S109D/T236A/E372G/1391L;
Q70E/S109D/T236A/E372G/V434S; Q70E/S109D/T236L/E372G/S386K/S404Y;
Q70E/S109D/E372G; Q70E/S109D/E372G/G398E; Q70E/S109D/E372G/V431E;
Q70E/T236A/E372G; Q70E/T236A/E372G/G398E;
Q70E/T236A/R281L/A325S/E372G; Q70E/T236L/E372G/G398E;
Q70E/E372G/V434S; Q70E/E372G/G398E/V434S;
S71L/M199A/K233C/E372G/K407L; S71L/E372G; K78R/Y182C/G356A/E372G;
K78R/V247A/L248S/G356A/E372G; K78R/V247A/E372G; K78R/G356A/E372G;
K78R/E372G/K399E/R423G; K78R/D373G; S109D/T236A/R281L/E372G;
S109D/T236L/R281L/A325R/E372G; S109D/R281L/E372G;
Q116C/M199A/K233C/E372G/K407L; Q116C/M199A/F360M/E372G;
K127R/G356A/E372G; K127R/E372G/D373G; Y182C/V247A/G356A;
L201H/E372G; M206T/L248S/H274R/K399E; M206T/L248S/E372G;
K211H/E372G/K407E; K233C/F360M/E372G/V380W/Q424H;
K233C/E372G/V380W; K233C/E372G/K407L;
K223T/K237A/E372G/K399T/K407E; V247A/L248S/G356A/E372G;
R281L/A325S/E372G/A397S; R284H/E372G; T329S/N330H/E372G/R375V;
N330Y/E372G/F436G; G356A/E372G; G356A/E372G/K399E/R423G;
G356A/D373G; F360M/E372G/Q424H; I371G/E372G/N433A; E372G/K378D;
E372G/K378D/K399T/K407E; E372G/I391L/S404Y/V434S; E372G/K399T;
E372G/K399T/K407E; E372G/K407E; E372G/K407R; and/or E372G/L435S6,
wherein the positions are numbered by correspondence with the amino
acid sequence set forth in SEQ ID NO:2.
In some additional embodiments, the nucleic acid constructs
provided herein further comprise a genetic element that facilitates
stable integration into a fungal host genome. In some embodiments,
the genetic element facilitates integration into a fungal host
genome by homologous recombination. In some additional embodiments,
the nucleic acid constructs comprise a fungal origin of
replication. In some embodiments, the fungal origin of replication
is a yeast origin of replication. In some additional embodiments,
the polynucleotide sequence of the nucleic acid constructs are
operatively linked to a promoter sequence that is functional in a
fungal cell. In some embodiments, the promoter sequence is a fungal
promoter sequence. In some further embodiments, the fungal promoter
sequence is a yeast promoter sequence. In some embodiments, the
polynucleotide sequence of the nucleic acid constructs are
operatively linked to a transcription termination sequence that is
functional in a fungal cell. In some additional embodiments, the
polynucleotide sequences of the nucleic acid constructs contain
codons optimized for expression in a yeast cell.
The present invention also provides recombinant fungal host cells
comprising a polynucleotide sequence that encodes a polypeptide
which is capable of catalyzing the isomerization of D-xylose
directly to D-xylulose, wherein the polynucleotide is selected
from: (a) a polynucleotide that encodes a polypeptide comprising an
amino acid sequence having at least 70% identity to SEQ ID NO:2,
and (b) a polynucleotide that hybridizes under stringent
hybridization conditions to the complement of a polynucleotide that
encodes a polypeptide having the amino acid sequence of SEQ ID
NO:2. In some embodiments, the polynucleotide sequence is a
polynucleotide sequence of any of the nucleic acid constructs
provided herein. In some embodiments, the polynucleotide is
integrated into the host cell genome. In some additional
embodiments, the host cell is a yeast cell. In some further
embodiments, the host cell has had one or more native genes deleted
from its genome. In some embodiments, the deletion results in one
or more phenotypes selected from increased transport of xylose into
the host cell, increased xylulose kinase activity, increased flux
through the pentose phosphate pathway, decreased sensitivity to
catabolite repression, increased tolerance to ethanol, increased
tolerance to acetate, increased tolerance to increased osmolarity,
increased tolerance to low pH, and reduced production of by
products, wherein comparison is made with respect to the
corresponding host cell without the deletion(s). In some additional
embodiments, the host cell is altered to overexpress one or more
polynucleotides. In some further embodiments, overexpression
results in one or more phenotypes selected from increased transport
of xylose into the host cell, increased xylulose kinase activity,
increased flux through the pentose phosphate pathway, decreased
sensitivity to catabolite repression, increased tolerance to
ethanol, increased tolerance to acetate, increased tolerance to
increased osmolarity, increased tolerance to low pH, and reduced
product of by products, wherein comparison is made to the
corresponding unaltered host cell. In some further embodiments, the
host cell is capable of growth in a xylose-based culture medium. In
some additional embodiments, the host cell is capable of growth at
a rate of at least about 0.2 per hour in a xylose-based culture
medium. In some embodiments, the host cell is capable of
fermentation in a xylose-based culture medium. In some additional
embodiments, the host cell is capable of fermenting xylose in a
xylose-based culture medium. In some embodiments, the host cell is
capable of fermenting xylose at a rate of at least about 1 g/L/h in
a xylose-based culture medium. In some embodiments, the host cell
is capable of faster growth in a xylose-based culture medium as
compared to wild-type Saccharomyces cerevisiae. In some further
embodiments, the xylose-based culture medium is selected from a
product from a cellulosic saccharification process or a
hemicellulosic feedstock.
The present invention also provides processes for producing a
fermentation product, wherein the method comprises: providing the
recombinant host cells as provided herein, a fermentation medium
comprising xylose; and contacting the fermentation medium with the
recombinant fungal host cells under conditions suitable for
generating the fermentation product. In some embodiments, the
processes further comprise the step of recovering the fermentation
product. In some further embodiments, the fermenting step is
carried out under microaerobic or aerobic conditions. In some
embodiments, the fermenting step is carried out under anaerobic
conditions. In some additional embodiments, the fermentation
product is at least one alcohol, fatty acid, lactic acid, acetic
acid, 3-hydroxypropionic acid, acrylic acid, succinic acid, citric
acid, malic acid, fumaric acid, succinic acid, an amino acid,
1,3-propanediol, ethylene, glycerol, and/or a .beta.-lactam. In
some further embodiments, the alcohol is ethanol, butanol, and/or a
fatty alcohol. In some embodiments, the fermentation product is
ethanol. In some still further embodiments, the fermentation
product is a fatty alcohol that is a C8-C20 fatty alcohol. In some
additional embodiments, the fermentation medium comprises product
from a saccharification process.
DESCRIPTION OF THE FIGURES
FIG. 1 depicts the two pathways for converting D-xylose to
D-xylulose. In one pathway, the D-xylose can be converted to
xylitol by xylose reductase (3) or aldoreductase (4). The xylitol
can be further converted to D-xylulose with a xylulose reductase
(5). In the second pathway, D-xylose is converted directly to
D-xylulose with a xylose isomerase (1). The D-xylulose produced
from either pathway--can be further converted to D-xylulose-5-P
with a xylulokinase (2). The numbers in the figure correspond to
the numbers in this description.
FIGS. 2A-C depict the metabolic pathways for converting
D-xylulose-5-P to ethanol.
FIG. 2A depicts the pentose phosphate pathway (PPP). The substrates
and products are shown. The enzymes are represented by numbers as
follows: 6. Ribulose-5-phosphate 3-epimerase; 7. Transketolase
(TKL1); 8. Transaldolase (TAL1); 9. Ribose-5-phosphate
ketoisomerase (RKI1); 10. 6-phosphogluconate dehydrogenase (GND1);
11. 6-phosphogluconalactonase (SOL3); and 12.
Glucose-6-phosphate-1-dehydrogenase (ZWF).
FIG. 2B depicts the pathway of glycolysis. The substrates and
products are shown. The enzymes are represented by numbers as
follows: 13. Hexokinase; 14. Phosphoglucose isomerase; 15.
Phosphofructokinase; 16. Aldolase; 17. Triose phosphate isomerase;
18. Glyceraldehyde 3-phosphate dehydrogenase; 19.
3-Phosphoglycerate kinase; 20. Phosphoglyceromutase; 21. Enolase;
and 22. Pyruvate kinase.
FIG. 2C depicts the metabolic pathway for converting pyruvate to
ethanol. The substrates and products are shown. The enzymes are
represented by numbers as follows: 23. Pyruvate decarboxylase; 24.
Aldehyde dehydrogenase; and 25. Alcohol dehydrogenase.
FIG. 3 depicts the native Ruminococcus flavefaciens xylose
isomerase gene (SEQ ID NO:1).
FIG. 4 depicts the Ruminococcus flavefaciens xylose isomerase (SEQ
ID NO:2) encoded by the polynucleotide sequence depicted in FIG. 3
(SEQ ID NO:1).
FIG. 5 depicts a polynucleotide sequence (SEQ ID NO:3) that has
been codon optimized for expression in Saccharomyces cerevisiae.
This codon optimized polynucleotide sequence also encodes the
Ruminococcus flavefaciens xylose isomerase amino acid sequence of
SEQ ID NO:2.
FIG. 6 depicts vector PLS4420 which is an 8259 by vector having a 2
micron origin of replication, pBS (pBluescript) origin of
replication, a TEF1 promoter, a CYC1 terminator, a kanamycin
resistance gene, and an ampicillin resistance gene.
FIG. 7 provides a plot of Absorbance Units versus time, where
absorbance correlates to cell growth. The plot provides a
comparison of cell growth on xylose of two Saccharomyces cerevisiae
cell lines, NRRL YB-1952 (ARS culture collection) and S. cerevisiae
Superstart LYCC6469 (Lallemand Ethanol Collection), each
transformed with three different plasmids: 1. PLS1567, which is the
vector control (no xylose isomerase gene); 2. PLS1569, which
contains the codon-optimized xylose isomerase gene from Clostridium
phytofermentans, SEQ ID NO: 16; and 3. PLS4420, which contains
codon-optimized xylose isomerase gene from Ruminococcus
flavefaciens. The corresponding experiment is described in Example
3.
FIG. 8 provides the xylose consumed during fermentation for
Saccharomyces cerevisiae cell lines, NRRL YB-1952 (ARS culture
collection) and BY4741 each transformed with three different
plasmids. 1. PLS1567, which is the vector control (no xylose
isomerase gene); 2. PLS1569, which contains the codon-optimized
xylose isomerase gene from Clostridium phytofermentans, SEQ ID NO:
16; and 3. PLS4420, which contains codon-optimized xylose isomerase
gene from Ruminococcus flavefaciens. The corresponding experiment
is described in Example 5.
DESCRIPTION OF THE INVENTION
The present invention provides methods and compositions suitable
for use in the isomerization of xylose to xylulose.
All patents and publications, including all sequences disclosed
within such patents and publications, referred to herein are
expressly incorporated by reference. Unless otherwise indicated,
the practice of the present invention involves conventional
techniques commonly used in molecular biology, fermentation,
microbiology, and related fields, which are known to those of skill
in the art. Unless defined otherwise herein, all technical and
scientific terms used herein have the same meaning as commonly
understood by one of ordinary skill in the art to which this
invention belongs. Although any methods and materials similar or
equivalent to those described herein can be used in the practice or
testing of the present invention, the preferred methods and
materials are described. Indeed, it is intended that the present
invention not be limited to the particular methodology, protocols,
and reagents described herein, as these may vary, depending upon
the context in which they are used. The headings provided herein
are not limitations of the various aspects or embodiments of the
present invention.
Nonetheless, in order to facilitate understanding of the present
invention, a number of terms are defined below. Numeric ranges are
inclusive of the numbers defining the range. Thus, every numerical
range disclosed herein is intended to encompass every narrower
numerical range that falls within such broader numerical range, as
if such narrower numerical ranges were all expressly written
herein. It is also intended that every maximum (or minimum)
numerical limitation disclosed herein includes every lower (or
higher) numerical limitation, as if such lower (or higher)
numerical limitations were expressly written herein.
As used herein, the term "comprising" and its cognates are used in
their inclusive sense (i.e., equivalent to the term "including" and
its corresponding cognates).
As used herein and in the appended claims, the singular "a", "an"
and "the" include the plural reference unless the context clearly
dictates otherwise. Thus, for example, reference to a "host cell"
includes a plurality of such host cells.
Unless otherwise indicated, nucleic acids are written left to right
in 5' to 3' orientation; amino acid sequences are written left to
right in amino to carboxy orientation, respectively. The headings
provided herein are not limitations of the various aspects or
embodiments of the invention that can be had by reference to the
specification as a whole. Accordingly, the terms defined below are
more fully defined by reference to the specification as a
whole.
As used herein, the terms "isolated" and "purified" are used to
refer to a molecule (e.g., an isolated nucleic acid, polypeptide,
etc.) or other component that is removed from at least one other
component with which it is naturally associated.
As used herein, the term "recombinant" refers to a polynucleotide
or polypeptide that does not naturally occur in a host cell. A
recombinant molecule may contain two or more naturally-occurring
sequences that are linked together in a way that does not occur
naturally. A recombinant cell contains a recombinant polynucleotide
or polypeptide.
As used herein, the term "overexpress" is intended to encompass
increasing the expression of a protein to a level greater than the
cell normally produces. It is intended that the term encompass
overexpression of endogenous, as well as heterologous proteins.
For clarity, reference to a cell of a particular strain refers to a
parental cell of the strain as well as progeny and genetically
modified derivatives of the same. Genetically modified derivatives
of a parental cell include progeny cells that contain a modified
genome or episomal plasmids that confer for example, antibiotic
resistance, improved fermentation, the ability to utilize xylose as
a carbon source, etc.
A nucleic acid construct, nucleic acid (e.g., a polynucleotide),
polypeptide, or host cell is referred to herein as "recombinant"
when it is non-naturally occurring, artificial or engineered.
The terms "xylose isomerase" and "xylose isomerase polypeptide" are
used interchangeably herein to refer to an enzyme that is capable
of catalyzing the isomerization of D-xylose directly to D-xylulose.
The ability to catalyze the isomerization of D-xylose directly to
D-xylulose is referred to herein as "xylose isomerase activity". An
exemplary assay for detecting xylose isomerase activity is provided
in Example 2. The terms "protein" and "polypeptide" are used
interchangeably herein to refer to a polymer of amino acid
residues. The term "xylose isomerase polynucleotide" refers to a
polynucleotide that encodes a xylose isomerase polypeptide.
In some embodiments, xylose isomerase polynucleotides employed in
the practice of the present invention encode a polypeptide
comprising an amino acid sequence that is at least about 71%
identical, at least about 72% identical, at least about 73%
identical, at least about 74% identical, at least about 75%
identical, at least about 76% identical, at least about 77%
identical, at least about 78% identical, at least about 79%
identical, at least about 80% identical, at least about 81%
identical, at least about 82% identical, at least about 83%
identical, at least about 84% identical, at least about 85%
identical, at least about 86% identical, at least about 87%
identical, at least about 88% identical, at least about 89%
identical, at least about 90% identical, at least about 91%
identical, at least about 92% identical, at least about 93%
identical, at least about 94% identical, at least about 95%
identical, at least about 96% identical, at least about 97%
identical, at least about 98% identical, or at least about 99%
identical to SEQ ID NO: 2. In some embodiments, the xylose
isomerase polynucleotide encodes a polypeptide having an amino acid
sequence that consists of the sequence of SEQ ID NO: 2.
In some embodiments, xylose isomerase polynucleotides employed in
the practice of the present invention comprise a polynucleotide
sequence that is at least about 70% identical, at least about 71%
identical, at least about 72% identical, at least about 73%
identical, at least about 74% identical, at least about 75%
identical, at least about 76% identical, at least about 77%
identical, at least about 78% identical, at least about 79%
identical, at least about 80% identical, at least about 81%
identical, at least about 82% identical, at least about 83%
identical, at least about 84% identical, at least about 85%
identical, at least about 86% identical, at last about 87%
identical, at least about 88% identical, at least about 89%
identical, at least about 90% identical, at least about 91%
identical, at least about 92% identical, at least about 93%
identical, at least about 94% identical, at least about 95%
identical, at least about 96% identical, at least about 97%
identical, at least about 98% identical, or at least about 99%
identical to SEQ ID NO:1 or SEQ ID NO:3. In some embodiments, the
xylose isomerase polynucleotide comprises the polynucleotide
sequence of SEQ ID NO:1 or SEQ ID NO:3.
The terms "percent identity," "% identity", "percent identical,"
and "% identical," are used interchangeably herein to refer to the
percent amino acid or polynucleotide sequence identity that is
obtained by ClustalW analysis (version W 1.8 available from
European Bioinformatics Institute, Cambridge, UK), counting the
number of identical matches in the alignment and dividing such
number of identical matches by the length of the reference
sequence, and using the following ClustalW parameters to achieve
slow/accurate pairwise optimal alignments--DNA/Protein Gap Open
Penalty: 15/10; DNA/Protein Gap Extension Penalty: 6.66/0.1;
Protein weight matrix: Gonnet series; DNA weight matrix: Identity;
Toggle Slow/Fast pairwise alignments=SLOW or FULL Alignment;
DNA/Protein Number of K-tuple matches: 2/1; DNA/Protein number of
best diagonals: 4/5; DNA/Protein Window size: 4/5.
Two sequences are "aligned" when they are aligned for similarity
scoring using a defined amino acid substitution matrix (e.g.,
BLOSUM62), gap existence penalty and gap extension penalty so as to
arrive at the highest score possible for that pair of sequences
Amino acid substitution matrices and their use in quantifying the
similarity between two sequences are well known in the art (See,
e.g., Dayhoff et al., in Dayhoff [ed.], Atlas of Protein Sequence
and Structure," Vol. 5, Suppl. 3, Natl. Biomed. Res. Round.,
Washington D.C. [1978]; pp. 345-352; and Henikoff et al., Proc.
Natl. Acad. Sci. USA, 89:10915-10919 [1992], both of which are
incorporated herein by reference). The BLOSUM62 matrix is often
used as a default scoring substitution matrix in sequence alignment
protocols such as Gapped BLAST 2.0. The BLOSUM62 matrix is often
used as a default scoring substitution matrix in sequence alignment
protocols such as Gapped BLAST 2.0. The gap existence penalty is
imposed for the introduction of a single amino acid gap in one of
the aligned sequences, and the gap extension penalty is imposed for
each additional empty amino acid position inserted into an already
opened gap. The alignment is defined by the amino acid position of
each sequence at which the alignment begins and ends, and
optionally by the insertion of a gap or multiple gaps in one or
both sequences so as to arrive at the highest possible score. While
optimal alignment and scoring can be accomplished manually, the
process is facilitated by the use of a computer-implemented
alignment algorithm (e.g., gapped BLAST 2.0; See, Altschul et al.,
Nucleic Acids Res., 25:3389-3402 [1997], which is incorporated
herein by reference), and made available to the public at the
National Center for Biotechnology Information Website). Optimal
alignments, including multiple alignments can be prepared using
readily available programs such as PSI-BLAST (See e.g, Altschul et
al., supra).
The present invention also provides a recombinant nucleic acid
construct comprising a xylose isomerase polynucleotide sequence
that hybridizes under stringent hybridization conditions to the
complement of a polynucleotide which encodes a polypeptide having
the amino acid sequence of SEQ ID NO:2, wherein the polypeptide is
capable of catalyzing the isomerization of D-xylose directly to
D-xylulose. An exemplary polynucleotide sequence that encodes a
polypeptide having the amino acid sequence of SEQ ID NO:2 is
selected from the group consisting of SEQ ID NO:1 and SEQ ID
NO:3.
In some embodiments, the polynucleotide that hybridizes to the
complement of a polynucleotide which encodes a polypeptide having
the amino acid sequence of SEQ ID NO:2, does so under high or very
high stringency conditions to the complement of a reference
sequence encoding a polypeptide having the sequence of SEQ ID NO:2
(e.g., over substantially the entire length of the reference
sequence).
Nucleic acids "hybridize" when they associate, typically in
solution. There are numerous texts and other reference materials
that provide details regarding hybridization methods for nucleic
acids (See e.g., Tijssen, Laboratory Techniques in Biochemistry and
Molecular Biology-Hybridization with Nucleic Acid Probes," Part 1,
Chapter 2, Elsevier, New York, [1993], incorporated herein by
reference). For polynucleotides of at least 100 nucleotides in
length, low to very high stringency conditions are defined as
follows: prehybridization and hybridization at 42.degree. C. in
5.times.SSPE, 0.3% SDS, 200 mg/ml sheared and denatured salmon
sperm DNA, and either 25% formamide for low stringencies, 35%
formamide for medium and medium-high stringencies, or 50% formamide
for high and very high stringencies, following standard Southern
blotting procedures. For polynucleotides of at least 200
nucleotides in length, the carrier material is finally washed three
times each for 15 minutes using 2.times.SSC, 0.2% SDS at least at
50.degree. C. (low stringency), at least at 55.degree. C. (medium
stringency), at least at 60.degree. C. (medium-high stringency), at
least at 65.degree. C. (high stringency), and at least at
70.degree. C. (very high stringency).
The terms "corresponding to", "reference to" and "relative to" when
used in the context of the numbering of a given amino acid or
polynucleotide sequence refers to the numbering of the residues of
a specified reference sequence when the given amino acid or
polynucleotide sequence is compared to the reference sequence.
The "position" is denoted by a number that sequentially identifies
each amino acid in the reference sequence based on its position
relative to the N-terminus. Owing to deletions, insertions,
truncations, fusions, and the like that must be taken into account
when determining an optimal alignment, in general the amino acid
residue number in a test sequence determined by simply counting
from the N-terminal will not necessarily be the same as the number
of its corresponding position in the reference sequence. For
example, in a case where there is a deletion in an aligned test
sequence, there will be no amino acid that corresponds to a
position in the reference sequence at the site of deletion. Where
there is an insertion in an aligned reference sequence, that
insertion will not correspond to any amino acid position in the
reference sequence. In the case of truncations or fusions there can
be stretches of amino acids in either the reference or aligned
sequence that do not correspond to any amino acid in the
corresponding sequence.
As used herein, the term "by-product" refers to an organic molecule
that is an undesired product of a particular fermentation
process.
As used herein, the term "transformed" or "transformation" used in
reference to a cell means that the cell has a non-native nucleic
acid sequence integrated into its genome or has an episomal plasmid
that is maintained through multiple generations.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides methods and compositions suitable
for use in the isomerization of xylose to xylulose.
The initial metabolic pathways for xylose utilization in fungi and
bacteria differ. In most fungi, including xylose-fermenting yeasts
(e.g., Pichia stipitis, Pachysolen tannophilus, and Candida
shehatae), D-xylose is converted to D-xylulose by two
oxidoreductases involving cofactors NAD(P)H and NAD(P)+. (See,
Matsushika et al., Appl. Microbiol. Biotechnol., 84:37-53 [2009]).
In these organisms, D-xylose is initially reduced to xylitol by
NAD(P)H-dependent xylose reductase (XR) (EC 1.1.1.21). Xylitol is
subsequently oxidized to D-xylulose by NAD+-dependent xylitol
dehydrogenase (XDH) (EC 1.1.1.9). Xylulokinase (XK) (EC 2.7.1.17)
subsequently phosphorylates D-xylulose to produce D-xylulose
5-phosphate (X5P), which is then further metabolized through the
pentose phosphate pathway (PPP).
However, most strains of S. cerevisiae cannot utilize xylose even
though the genes encoding XR, XDH, and XK are present in its
genome, as the expression levels of these enzymes are too low to
allow xylose utilization (See, Matsushika et al., supra). Some
strains have been shown to natively utilize xylose but at very low
rates and fermentation to ethanol has not been detected (See,
Wenger et al., PLoS Genet., 6(5):e1000942 [2010]). Even when the
endogenous genes are overexpressed in S. cerevisiae, only slow
growth on xylose has been observed (See, Matsushika et al.,
supra).
In contrast, most bacteria (e.g., Escherichia coli and Streptomyces
species) can isomerize D-xylose directly to D-xylulose by using a
xylose isomerase (XI) (EC 5.3.1.5) (See, Matsushika et al., supra).
In bacteria, as in fungi, the D-xylulose is phosphorylated to
D-xylulose 5-phosphate by XK, which is then further metabolized
through the pentose phosphate pathway.
Efforts to express a functional heterologous xylose isomerase gene
(xylA) in S. cerevisiae and grow the yeast on xylose has met with
very limited success (See e.g., Matsushika et al. supra). It has
been reported that xylose isomerase genes from the fungi Piromyces
(Kuyper et al. FEMS Yeast Res., 4:69-78 [2003]) and Orpinomyces
(Madhaven et al., Appl. Microbiol. Biotechnol., 82:1067-1078
[2009a]) have been functionally expressed in S. cerevisiae, but
that growth on xylose was very slow. In addition, the functional
expression of the Thermus thermophilus xylose isomerase (Accession
No. 1BXB) in S. cerevisiae has been reported (See, Walfridsson et
al., Appl. Environ. Microbiol., 62:4648-4651 [1996]). The success
in producing an active xylose isomerase by expressing the T.
thermophilus xylA gene in S. cerevisiae may have been due to the
relatedness between the two organisms, as T. thermophilus diverged
from the domain of eubacteria and may, in many respects, be more
closely related to S. cerevisiae than are the eubacteria (Id., at
4651).
Heterologous expression of xylose isomerase genes from Actinoplanes
missouriensis and Clostridium thermosulfurogenes in S. cerevisiae
generated inactive proteins, even though their messenger RNA could
be detected (See, Amore et al., Appl. Microbiol. Biotechnol.,
30:351-357 [1989]); and Moes et al., Biotech. Left., 18:269-274
[1996]; and Matsushika et al., supra). Other studies report the
heterologous expression of the xylA from E. coli (See e.g., Sarthy
et al., Appl. Environ. Microbiol., 53:1996-2000 [1987]), Bacillus
subtilis (Amore et al., Appl. Microbiol. Biotechnol., 30:351-357
[1989]), and Streptomyces rubiginosus (Gardonyi et al., Enzyme
Microb. Technol., 32:252-259 [2003]) in S. cerevisiae resulted in
mainly insoluble proteins which were catalytically inactive (See,
Matsushika et al., supra). In addition, some reports indicate that
attempts to produce xylose isomerase from recombinant S. cerevisiae
transformed with the xylA genes from Bacillus subtilis and
Lactobacillus pentosus resulted in inactive protein (See,
Walfridsson et al., supra).
In further studies, the results of screening for xylose isomerase
activity in S. cerevisiae transformed with the xylose isomerase
genes from various organisms have been reported (See e.g., Brat et
al., Appl. Environ. Microbiol. Doi:10.1128/AEM.02522-9 [13 Feb.
2009]). The xylose isomerases are reported to have from 17% to 60%
sequence identity to the xylose isomerase from Piromyces. While
transformants expressing the xylose isomerase from Clostridium
phytofermentans (DSM 18823) could grow on xylose medium, S.
cerevisiae transformed with the xylose isomerase gene from the
following organisms could not: Bacillus licheniformis (DSM 13),
Burkholderia xenovaorans (DSM 17367), Lactobacillus pentosus (DSM
20314), Leifsonia xyli subsp. cynodontis (DSM 46306), Pseudomonas
savastanoi pvar. Phaseolicola (DSM 50282), Robiginitalea biformata
(DSM 15991), Saccharophagus degradans (DSM 17024), Staphylococcus
xylosus (DSM 20266), Streptomyces diastaticus subsp. diastaticus
(DSM 40496), Xanthomonas campestris pvar. campestris (DSM 3586),
Salmonella typhimurium (71-098L), Agrobacterium tumefaciens, and
Arabidopsis thaliana (See, Brat et al., supra).
The present invention provides sequences that are capable of
conferring the property of xylose-utilization in a non-mammalian,
eukaryotic host cell, such as, for example, a fungal host cell.
This biological sequence and variants thereof, encode xylose
isomerases, which catalyze the isomerization of D-xylose directly
to D-xylulose, as depicted in FIG. 1. Xylose isomerase is
distinguished from xylose reductase (XD), which catalyzes the
conversion of xylose to xylitol. Xylose isomerase is also
distinguished from xylitol dehydrogenase (XD), which catalyzes the
conversion of xylitol to D-xylulose (See, FIG. 1).
Xylose utilization by these host cells results in useful products
that are produced metabolically by the host cell. In these host
cells, D-xylulose may be phosphorylated by a native or recombinant
xylulokinase to xylulose-5-P, as depicted in FIG. 1. The
xylulose-5-P may be further metabolized by enzymes in the pentose
phosphate pathway to products such as glucose-6-P, fructose-6-P,
glyceraldehydes-3-P, and the like. The pentose phosphate pathway
and relevant enzymes and products are depicted in FIG. 2A. As used
herein, the terms "enzyme from the pentose phosphate pathway" and
"pentose phosphate pathway enzyme" are used interchangeably to
refer to an enzyme from the group of enzymes involved in the
pentose phosphate pathway, (i.e., 6. Ribulose-5-phosphate
ketoisomerase (RK11); 7. Transketolase (TKL1); 8. Transaldolase
(TAL1); 9. Ribose-5-phosphate ketoisomerase (RK11); 10.
6-phosphogluconate dehydrogenase (GND1); 11.
6-phosphogluconalactonase (SOL3); and/or 12.
Glucose-6-phosphate-1-dehydrogenase (ZWF); the reference numbers
are depicted in FIG. 2A).
Products of the pentose phosphate pathway may be further
metabolized through the process of glycolysis. The metabolic
process of glycolysis is depicted in FIG. 2B. As used herein, the
term "glycolytic enzyme" refers to an enzyme from the group of
enzymes involved in glycolysis (i.e.: 13. Hexokinase; 14.
Phosphoglucose isomerase; 15. Phosphofructokinase; 16. Aldolase;
17. Triose phosphate isomerase; 18. Glyceraldehyde phosphate
dehydrogenase; 19. Phosphoglycerate kinase; 20.
Phosphoglyceromutase; 21. Enolase; and/or 22. Pyruvate kinase; the
reference numbers are depicted in FIG. 2B).
Pyruvate from the glycolytic pathway (i.e., glycolysis) may be
further metabolized to ethanol as shown in FIG. 2C by ethanologenic
enzymes. As used herein, the term "ethanologenic enzyme" refers to
an enzyme involved in the conversion of pyruvate to ethanol, (e.g.,
a pyruvate decarboxylase, an aldehyde dehydrogenase, and/or an
alcohol dehydrogenase). The term "ethanologenic pathway" refers to
the pathway depicted in FIG. 2C.
Therefore, the polynucleotide sequences described herein are useful
for creating recombinant fungal host cells, particularly yeast host
cells, that are capable of isomerizing D-xylose directly to
D-xylulose, which can lead to the production of desirable
fermentation products (e.g., an alcohol, such as ethanol, butanol,
and the like, including a fatty alcohol [such as a C8-C20 fatty
alcohol], a fatty acid [e.g., a C8-C20 fatty acid], lactic acid,
3-hydroxypropionic acid, acrylic acid, acetic acid, succinic acid,
citric acid, malic acid, fumaric acid, an amino acid,
1,3-propanediol, ethylene, glycerol, a .beta.-lactam, and the
like).
Recombinant Nucleic Acid Constructs
The present invention provides a recombinant nucleic acid construct
comprising a polynucleotide sequence that encodes a polypeptide
comprising an amino acid sequence having at least 70% identity to
SEQ ID NO: 2, wherein the polypeptide is capable of catalyzing the
isomerization of D-xylose directly to D-xylulose. SEQ ID NO: 2
corresponds to the amino acid sequence encoding a putative xylose
isomerase from the bacteria, Ruminococcus flavefaciens (See, FIG.
4). SEQ ID NO: 1 corresponds to the native R. flavefaciens
polynucleotide sequence that encodes the putative R. flavefaciens
xylose isomerase (SEQ ID NO: 2), both of which are provided
below.
TABLE-US-00001 (SEQ ID NO: 1)
ATGGAATTTTTCAGCAATATCGGTAAAATTCAGTATCAGGGACCAAAAAG
TACTGATCCTCTCTCATTTAAGTACTATAACCCTGAAGAAGTCATCAACG
GAAAGACAATGCGCGAGCATCTGAAGTTCGCTCTTTCATGGTGGCACACA
ATGGGCGGCGACGGAACAGATATGTTCGGCTGCGGCACAACAGACAAGAC
CTGGGGACAGTCCGATCCCGCTGCAAGAGCAAAGGCTAAGGTTGACGCAG
CATTCGAGATCATGGATAAGCTCTCCATTGACTACTATTGTTTCCACGAT
CGCGATCTTTCTCCCGAGTATGGCAGCCTCAAGGCTACCAACGATCAGCT
TGACATAGTTACAGACTATATCAAGGAGAAGCAGGGCGACAAGTTCAAGT
GCCTCTGGGGTACAGCAAAGTGCTTCGATCATCCAAGATTCATGCACGGT
GCAGGTACATCTCCTTCTGCTGATGTATTCGCTTTCTCAGCTGCTCAGAT
CAAGAAGGCTCTCGAGTCAACAGTAAAGCTCGGCGGTAACGGTTACGTTT
TCTGGGGCGGACGTGAAGGCTATGAGACACTTCTTAATACAAATATGGGA
CTCGAACTCGACAATATGGCTCGTCTTATGAAGATGGCTGTTGAGTATGG
ACGTTCGATCGGCTTCAAGGGCGACTTCTATATCGAGCCCAAGCCCAAGG
AGCCCACAAAGCATCAGTACGATTTCGATACAGCTACTGTTCTGGGATTC
CTCAGAAAGTACGGTCTCGATAAGGATTTCAAGATGAATATCGAAGCTAA
CCACGCTACACTTGCTCAGCATACATTCCAGCATGAGCTCCGTGTTGCAA
GAGACAATGGTGTGTTCGGTTCTATCGACGCAAACCAGGGCGACGTTCTT
CTTGGATGGGATACAGACCAGTTCCCCACAAATATCTACGATACAACAAT
GTGTATGTATGAAGTTATCAAGGCAGGCGGCTTCACAAACGGCGGTCTCA
ACTTCGACGCTAAGGCACGCAGAGGGAGCTTCACTCCCGAGGATATCTTC
TACAGCTATATCGCAGGTATGGATGCATTTGCTCTGGGCTTCAGAGCTGC
TCTCAAGCTTATCGAAGACGGACGTATCGACAAGTTCGTTGCTGACAGAT
ACGCTTCATGGAATACCGGTATCGGTGCAGACATAATCGCAGGTAAGGCA
GATTTCGCATCTCTTGAAAAGTATGCTCTTGAAAAGGGCGAGGTTACAGC
TTCACTCTCAAGCGGCAGACAGGAAATGCTGGAGTCTATCGTAAATAACG
TTCTTTTCAGTCTGTAA (SEQ ID NO: 2)
MEFFSNIGKIQYQGPKSTDPLSFKYYNPEEVINGKTMREHLKFALSWWHT
MGGDGTDMFGCGTTDKTWGQSDPAARAKAKVDAAFEIMDKLSIDYYCFHD
RDLSPEYGSLKATNDQLDIVTDYIKEKQGDKFKCLWGTAKCFDHPRFMHG
AGTSPSADVFAFSAAQIKKALESTVKLGGNGYVFWGGREGYETLLNTNMG
LELDNMARLMKMAVEYGRSIGFKGDFYIEPKPKEPTKHQYDFDTATVLGF
LRKYGLDKDFKMNIEANHATLAQHTFQHELRVARDNGVFGSIDANQGDVL
LGWDTDQFPTNIYDTTMCMYEVIKAGGFTNGGLNFDAKARRGSFTPEDIF
YSYIAGMDAFALGFRAALKLIEDGRIDKFVADRYASWNTGIGADIIAGKA
DFASLEKYALEKGEVTASLSSGRQEMLESIVNNVLFSL
In some embodiments, recombinant nucleic acid constructs of the
present invention further comprise a polynucleotide sequence
(genetic) element that facilitates integration into a fungal host
cell genome, by homologous or non-homologous recombination. In some
embodiments, the nucleic acid construct of the present invention
further comprises an origin of replication that is functional in a
fungal cell (e.g., a yeast origin of replication). Typically, the
fungal host cell is a yeast or filamentous fungal cell, more
typically, a yeast cell. In some embodiments, nucleic acid
constructs of the present invention comprise a transcriptional
regulatory element that is functional in a fungal cell. For
example, in some embodiments the recombinant nucleic acid construct
comprises a promoter sequence and/or transcription terminator
sequence that is functional in a fungal cell such that the xylose
isomerase polynucleotide is operatively linked to the promoter
sequence and/or transcription terminator sequences.
Xylose isomerase polynucleotides that are suitable for use in the
practice of the present invention include those encoding variants
of SEQ ID NO: 2. These variants include those having amino acid
sequences with one or more conservative or non-conservative
substitutions relative to the amino acid sequence of SEQ ID NO: 2.
As used herein, the term "conservative substitution" refers to the
substitution of a residue for another residue that does not
generally alter the specific activity of the encoded polypeptide.
An exemplary conservative substitution is a substitution that is
within the same group of basic amino acids (arginine, lysine and
histidine), acidic amino acids (glutamic acid and aspartic acid),
polar amino acids (glutamine and asparagine), hydrophobic amino
acids (leucine, isoleucine and valine), aromatic amino acids
(phenylalanine, tryptophan and tyrosine), and small amino acids
(glycine, alanine, serine, threonine, proline, cysteine and
methionine) Amino acid substitutions that do not generally alter
the specific activity are known in the art and are described, for
example, by H. Neurath and R. L. Hill, 1979, in "The Proteins,"
Academic Press, New York, which is incorporated herein by
reference. The most commonly occurring exchanges are Ala/Ser,
Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr. Ser/Asn, Ala/Val,
Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val,
Ala/Glu, and Asp/Gly, as well as these in reverse.
Other xylose isomerase polynucleotides suitable for use in the
practice of the present invention include those encoding variants
of R. flavefaciens xylose isomerase generated by mutagenesis,
recombination, or other protein engineering method followed by
screening of the variants for xylose utilization using a method,
such as that described in Example 2. In some embodiments, the
resulting variants comprise one or more substitutions (conservative
or non-conservative), deletions, and/or insertions. The present
invention thus provides methods for making improved R. flavefaciens
xylose isomerase polynucleotide variants, wherein the method
comprises introducing one or more modifications into a
polynucleotide encoding SEQ ID NO:2 to produce a modified
polynucleotide, wherein the modification is selected from at least
one substitution, at least one deletion, and/or at least one
insertion; transforming a host cell with the modified
polynucleotide; and screening the transformed host cell for an
improvement in a desired phenotype relative to the corresponding
untransformed host cell. Exemplary phenotypes include improved
utilization of a pentose sugar (e.g., xylose, arabinose, etc.),
stability, specific activity, lower Ki for xylitol, ethanol/acetate
tolerance and/or tolerance to low pH, decreased by-product
formation, and/or increased ethanol yield. Exemplary desirable
xylose utilization phenotypes include the ability to ferment xylose
to ethanol, the ability to ferment xylose to other metabolic
intermediates/products, the ability to undergo aerobic or anaerobic
growth on xylose, and the like.
Methods for generating variant libraries of polynucleotides
encoding modified polypeptides are well known in the art. For
example, mutagenesis and directed evolution methods can be readily
applied to polynucleotides encoding the xylose isomerase
polypeptide of SEQ ID NO:2 to generate variant libraries that can
be expressed, screened, and assayed using the methods described
herein. Mutagenesis and directed evolution methods are well known
in the art (See e.g., Ling et al., Anal. Biochem., 254(2):157-78
[1997]; Dale et al., Meth. Mol. Biol., 57:369-74 [1996]; Smith,
Ann. Rev. Genet., 19:423-462 [1985]; Botstein et al., Science,
229:1193-1201 [1985]; Carter, Biochem. J., 237:1-7 [1986]; Kramer
et al., Cell, 38:879-887 [1984]; Wells et al., Gene, 34:315-323
[1985]; Minshull et al., Curr. Op. Chem. Biol., 3:284-290 [1999];
Christians et al., Nat. Biotechnol., 17:259-264 [1999]; Crameri et
al., Nature, 391:288-291 [1998]; Crameri, et al., Nat. Biotechnol.,
15:436-438 [1997]; Zhang et al., Proc. Nat. Acad. Sci. U.S.A.,
94:4504-4509 [1997]; Crameri et al., Nat. Biotechnol., 14:315-319
[1996]; Stemmer, Nature, 370:389-391 [1994]; Stemmer, Proc. Nat.
Acad. Sci. USA, 91:10747-10751 [1994]; WO 95/22625; WO 97/0078; WO
97/35966; WO 98/27230; WO 00/42651; WO 01/75767; and WO
2009/152336, all of which are incorporated herein by
reference).
In some embodiments, the present invention provides Ruminococcus
flavefaciens xylose isomerase polypeptide variants that comprise at
least one modification that is a substitution, insertion, and/or
deletion relative to SEQ ID NO:2. Typically, the polypeptide
variant has from about 1 to about 2, about 1 to about 3, about 4,
about 5, about 6, about 7, about 8, about 9, about 10, about 11,
about 12, about 13, about 14, about 15, about 16, about 17, about
18, about 19, about 20, about 21, about 22, about 23, about 24,
about 25, about 26, about 27, about 28, about 29, about 30, about
31, about 32, about 33, about 34, about 35, about 36, about 37,
about 38, about 39, about 40, about 41, about 42, about 43, about
44, about 45, about 46, about 47, about 48, about 49, up to about
50, about 75, about 100, or about 130 modifications.
In some embodiments, the xylose isomerase variants of the present
invention comprise glycine at a position corresponding to position
372 in SEQ ID NO:2, as determined by an alignment of the variant
with SEQ ID NO:2 (i.e., E372G; SEQ ID NO:19, which is encoded by
SEQ ID NO:18). These sequences are provided below.
TABLE-US-00002 (SEQ ID NO: 18)
ATGGAATTTTTCTCCAACATCGGAAAAATCCAATACCAAGGTCCAAAATC
CACAGATCCTTTGTCTTTTAAATATTATAATCCTGAAGAAGTAATCAACG
GTAAGACCATGAGGGAGCATTTGAAATTCGCTCTATCATGGTGGCACACA
ATGGGTGGCGATGGTACTGATATGTTCGGATGTGGTACTACGGACAAGAC
CTGGGGTCAATCCGACCCAGCGGCAAGAGCTAAGGCCAAAGTTGATGCTG
CTTTCGAAATTATGGATAAGCTGAGCATTGATTACTACTGCTTCCATGAT
AGAGACCTTTCTCCAGAATATGGCTCCTTGAAAGCGACCAATGATCAACT
GGACATTGTTACTGATTACATCAAGGAGAAGCAGGGCGATAAATTCAAGT
GTTTATGGGGCACTGCTAAATGCTTTGATCACCCCAGGTTCATGCACGGT
GCAGGAACTTCTCCTAGTGCCGATGTTTTCGCTTTTTCTGCTGCGCAAAT
AAAGAAAGCATTAGAATCTACCGTCAAGTTGGGCGGTAATGGTTATGTCT
TTTGGGGTGGTAGAGAAGGTTACGAGACCCTGCTGAATACTAACATGGGC
TTAGAACTGGACAACATGGCTAGGCTAATGAAGATGGCCGTAGAATACGG
TAGGTCTATTGGATTCAAAGGTGACTTCTACATCGAGCCTAAACCCAAGG
AACCTACTAAGCACCAGTACGACTTCGACACTGCTACCGTATTAGGTTTT
TTAAGGAAGTACGGGTTGGATAAAGACTTCAAAATGAACATCGAAGCCAA
TCACGCCACACTAGCACAACACACATTCCAGCATGAGTTACGTGTGGCTA
GGGATAACGGTGTATTCGGTTCTATTGATGCTAACCAAGGTGACGTATTG
TTAGGATGGGACACGGATCAATTCCCCACAAACATTTATGATACTACTAT
GTGTATGTATGAGGTCATTAAAGCCGGTGGTTTCACAAATGGCGGCCTGA
ACTTTGATGCGAAAGCTCGTAGGGGTTCATTCACGCCTGAAGATATTTTC
TATAGTTACATTGCTGGTATGGATGCTTTCGCGTTAGGGTTTAGAGCAGC
TCTTAAATTGATTGGAGACGGTAGAATTGACAAGTTTGTGGCGGATAGGT
ATGCATCTTGGAATACCGGTATTGGTGCAGATATTATTGCCGGAAAAGCC
GATTTTGCATCATTGGAAAAATATGCTTTGGAAAAAGGTGAAGTTACCGC
GTCATTGTCTTCAGGTAGGCAAGAGATGCTGGAATCTATTGTCAACAACG
TATTGTTTAGTTTGTAA (SEQ ID NO: 19)
MEFFSNIGKIQYQGPKSTDPLSFKYYNPEEVINGKTMREHLKFALSWWHT
MGGDGTDMFGCGTTDKTWGQSDPAARAKAKVDAAFEIMDKLSIDYYCFHD
RDLSPEYGSLKATNDQLDIVTDYIKEKQGDKFKCLWGTAKCFDHPRFMHG
AGTSPSADVFAFSAAQIKKALESTVKLGGNGYVFWGGREGYETLLNTNMG
LELDNMARLMKMAVEYGRSIGFKGDFYIEPKPKEPTKHQYDFDTATVLGF
LRKYGLDKDFKMNIEANHATLAQHTFQHELRVARDNGVFGSIDANQGDVL
LGWDTDQFPTNIYDTTMCMYEVIKAGGFTNGGLNFDAKARRGSFTPEDIF
YSYIAGMDAFALGFRAALKLIGDGRIDKFVADRYASWNTGIGADIIAGKA
DFASLEKYALEKGEVTASLSSGRQEMLESIVNNVLFSL
In some embodiments, the xylose isomerase polynucleotides referred
to herein encode polypeptides comprising an amino acid sequence
having the substitution E372G, wherein amino acid position is
determined by alignment with SEQ ID NO:2. An exemplary
polynucleotide encoding a E372G variant of the R. flavefaciens
xylose isomerase is provided as SEQ ID NO: 18.
Also suitable for use in the practice of the present invention are
polynucleotides encoding a truncated variant of Ruminococcus
flavefaciens xylose isomerase or sequence variant thereof that is
capable of catalyzing the isomerization of X-xylose directly to
D-xylulose. These truncation variants may be truncated at the
carboxy (C)-terminus and/or the amino (N)-terminus. Typically, the
truncation is from about 1 to about 50 amino acid residues
Those having ordinary skill in the art will understand that due to
the degeneracy of the genetic code, a multitude of nucleotide
sequences that encode the xylose isomerase polypeptides described
herein exist. Table 1 provides the standard triplet genetic code
for each amino acid. For example, the codons AGA, AGG, CGA, CGC,
CGG, and CGU all encode the amino acid arginine. Thus, at every
position in the nucleic acids referred to herein, where an arginine
is specified by a codon, the codon can be altered to any of the
corresponding codons described above without altering the encoded
polypeptide. It is understood that U in an RNA sequence corresponds
to T in a DNA sequence. The invention contemplates and provides
each and every possible variation of nucleic acid sequence encoding
a polypeptide of the invention that could be made by selecting
combinations based on possible codon choices.
TABLE-US-00003 TABLE 1 Genetic Code Amino Acids Codon Alanine Ala A
GCA GCC GCG GCU Cysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU
Glutamic Glu E GAA GAG acid Phenylala- Phe F UUC UUU nine Glycine
Gly G GGA GGC GGG GGU Histidine His H CAC CAU Isoleucine Ile I AUA
AUC AUU Lysine Lys K AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUU
Methionine Met M AUG Asparagine Asn N AAC AAU Proline Pro P CCA CCC
CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG
CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC
ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine
Tyr Y UAC UAU
A DNA sequence may also be designed for high codon usage bias
(i.e., codons that are used at higher frequency in the protein
coding regions than other codons that code for the same amino
acid). The preferred codons may be determined in relation to codon
usage in a single gene, a set of genes of common function or
origin, highly expressed genes, the codon frequency in the
aggregate protein coding regions of the whole organism, codon
frequency in the aggregate protein coding regions of related
organisms, or combinations thereof. Codons whose frequency
increases with the level of gene expression are typically optimal
codons for expression. In particular, a DNA sequence can be
optimized for expression in a particular host organism. References
providing preference information for a wide range of organisms are
readily available (See e.g., Henaut and Danchin in Neidhardt et al.
[eds.], Escherichia coli and Salmonella, ASM Press, Washington
D.C., [1987], p. 2047-2066, which is incorporated herein by
reference).
A variety of methods are known for determining the codon frequency
(e.g., codon usage, relative synonymous codon usage) and codon
preference in specific organisms, including multivariate analysis,
for example, using cluster analysis or correspondence analysis, and
the effective number of codons used in a gene (See, GCG
CodonPreference, Genetics Computer Group Wisconsin Package; Peden,
Codon W, University of Nottingham; McInerney, Bioinform., 14:372-73
[1998]; Stenico et al., Nucl. Acids Res. 222437-46 [1994]; Wright,
Gene 87:23-29 [1990]; Wada et al., Nucl. Acids Res., 20:2111-2118
[1992]; Nakamura et al., Nucl. Acids Res., 28:292 [2000]; and
Henaut and Danchin, supra; all of which are incorporated herein by
reference). The data source for obtaining codon usage may rely on
any available nucleotide sequence capable of coding for a protein.
These data sets include nucleic acid sequences actually known to
express proteins (e.g., complete protein coding sequences-CDS),
expressed sequence tags (ESTs), or predicted coding regions of
genomic sequences (See e.g., Mount, Bioinformatics: Sequence and
Genome Analysis, Chapter 8, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., [2001]; Uberbacher, Methods Enzymol.,
266:259-281 [1996]; and Tiwari et al., Comput. Appl. Biosci.
13:263-270 [1997]; all of which are incorporated herein by
reference).
In some embodiments, the xylose isomerase polynucleotide contains
codons optimized for expression in a fungal cell, particularly a
yeast cell. An exemplary codon optimized xylose isomerase
polynucleotide sequence is provided as SEQ ID NO:3 (FIG. 5) which
exhibits improved expression in Saccharomyces cerevisiae as
compared to the wild-type sequence (SEQ ID NO:1).
Some silent mutations have been identified in R. flavefaciens
xylose isomerase polynucleotide variants. These silent mutations
include: t1263a, a1269g, and t1266g (where nucleotide position is
determined by alignment with SEQ ID NO:3). These silent mutations
correspond to the following codons: TCA encoding serine at position
421; GGG encoding glycine at position 422; and AGG encoding
arginine at position 423. The amino acid position referred to above
is the corresponding amino acid position in SEQ ID NO: 2. The
sequences are shown in SEQ ID NO:18 (nucleic acid) and SEQ ID NO:19
(amino acid)
In some embodiments, the xylose isomerase polynucleotides are
employed in recombinant nucleic acid constructs that comprise a
vector (e.g., a plasmid, a cosmid, a phage, a virus, a yeast
artificial chromosome (YAC), and the like), into which a xylose
isomerase polynucleotide sequence has been inserted. The xylose
isomerase polynucleotides provided herein find use when
incorporated into any one of a variety of vectors. Suitable vectors
include, but are not limited to chromosomal, nonchromosomal and
synthetic DNA sequences, yeast plasmids, vectors derived from
combinations of plasmids and phage DNA, and many others. Any
suitable vector that transduces genetic material into a cell, and,
if replication is desired, which is replicable and viable in the
relevant host find use in the present invention.
Nucleic acid constructs of the present invention find use in
transforming a host cell to permit the host to express the xylose
isomerase polypeptide. Methods for recombinant expression of
proteins in fungi are well known in the art, and a number of
vectors are available or can be constructed using routine methods
(See e.g., Zhu et al., Plasmid 6:128-33 [2009], incorporated herein
by reference; and the many standard reference works in this
field).
In some embodiments, recombinant nucleic acid constructs of the
present invention further comprise a transcriptional regulatory
element that is functional in a fungal cell. In some embodiments,
the nucleic acid construct comprises the xylose isomerase
polynucleotide operatively linked to a transcriptional regulatory
sequence (e.g., a promoter, transcription termination sequence, and
the like), that is functional in a fungal cell. Examples of
promoters that are functional in a fungal host cell include, but
are not limited to promoters from yeast and filamentous fungi.
Promoters that are suitable for use in the practice of the present
invention include endogenous or heterologous promoters and include
both constitutive and inducible promoters that are natural or
modified. Particularly useful promoters are those that are
insensitive to catabolite (glucose) repression and/or do not
require xylose for induction. Such promoters are well known in the
art. In some embodiments, a promoter sequence is operably linked to
the 5' region of the xylose isomerase coding sequence using routine
methods that are well known in the art.
Promoters that are suitable for use in the practice of the present
invention include, but are not limited to yeast promoters from
glycolytic genes (e.g., yeast phosphofructokinase (PFK), triose
phosphate isomerase (TPI), glyceraldehyde-3-phosphate dehydrogenase
(GPD, TDH3 or GAPDH), pyruvate kinase (PYK), phosphoglycerate
kinase (PGK) promoters, and the like; See e.g., WO 93/03159, which
is incorporated herein by reference); promoters of glucose
transporters; ribosomal protein encoding gene promoters; alcohol
dehydrogenase promoters (e.g., ADH1, ADH4, and the like), and the
enolase promoter (ENO).
Exemplary promoters that are useful for directing the transcription
of the nucleic acid constructs of the present invention in yeast
host cells include, but are not limited to those from the genes for
Saccharomyces cerevisiae enolase (eno-1), Saccharomyces cerevisiae
galactokinase (gall), Saccharomyces cerevisiae alcohol
dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase
(ADH1/ADH2/GAP), and Saccharomyces cerevisiae 3-phosphoglycerate
kinase, Saccharomyces cerevisiae transcription elongation factor
(TEF), Saccharomyces cerevisiae fructose 1,6-bisphosphate aldolase
(FBA1), and Saccharomyces cerevisiae 3-phosphate glycerate kinase
(PGK1). Other useful promoters for yeast host cells are well known
in the art (See e.g., Romanos et al., Yeast 8:423-488 [1992], which
is incorporated herein by reference).
Suitable filamentous fungal promoters that are useful in the
practice of the present invention include, but are not limited to
promoters obtained from the genes for Aspergillus oryzae TAKA
amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger
neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,
Aspergillus niger or Aspergillus awamori glucoamylase (glaA),
Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease,
Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans
acetamidase, and Fusarium oxysporum trypsin-like protease (See
e.g., WO 96/00787, which is incorporated herein by reference), as
well as the NA2-tpi promoter (a hybrid of the promoters from the
genes for Aspergillus niger neutral alpha-amylase and Aspergillus
oryzae triose phosphate isomerase), promoters such as cbh1, cbh2,
egl1, egl2, pepA, hfb1, hfb2, xyn1, amy, and glaA (See, Nunberg et
al., Mol. Cell Biol., 4:2306-2315 [1984]; Boel et al., EMBO J.
3:1581-85 [1984]; and EP 0 137 280A, all of which are incorporated
herein by reference), and mutant, truncated, and hybrid promoters
thereof. Promoters associated with chitinase production in fungi
also find use in some embodiments (See e.g., Blaiseau and Lafay,
Gene 120:243-248 [1992] [filamentous fungus Aphanocladium album];
and Limon et al., Curr. Genet., 28:478-83 [1995] [Trichoderma
harzianum]; both of which are incorporated herein by
reference).
Any other suitable promoter sequence that drives expression in a
fungal host cell, particularly a yeast host cell finds use in the
present invention. Suitable promoter sequences can be identified
using well known methods. In one approach, a putative promoter
sequence is linked 5' to a sequence encoding a reporter protein,
the construct is transfected into the host cell and the level of
expression of the reporter is measured. Expression of the reporter
can be determined by measuring, for example, mRNA levels of the
reporter sequence, an enzymatic activity of the reporter protein,
or the amount of reporter protein produced. For example, promoter
activity may be determined by using the green fluorescent protein
as coding sequence (See, Henriksen et al., Microbiol., 145:729-34
[1999], which is incorporated herein by reference) or a lacZ
reporter gene (See, Punt et al., Gene, 197:189-93 [1997], which is
incorporated herein by reference). In some embodiments, functional
promoters are derived from naturally occurring promoter sequences
by directed evolution methods (See e.g., Wright et al., Hum. Gene
Ther., 16:881-892 [2005], which is incorporated herein by
reference).
Exemplary transcription termination sequences (terminators) that
are functional in a fungal host cell, include transcription
termination sequences from yeast and filamentous fungi, that are
well known in the art. In some embodiments, the transcription
termination sequence is from a yeast. Exemplary yeast transcription
termination sequences include, but are not limited to CYC1, ADH1t,
ADH2t, etc. In some embodiments, the nucleic acid constructs of the
present invention contain a ribosome binding site for translation
initiation. In some embodiments, the construct includes appropriate
sequences for amplifying expression (e.g., an enhancer). Such
elements are well known in the art and any suitable enhancers
and/or transcription termination sequences, and/or ribosome binding
sites find use in the present invention.
In some additional embodiments, nucleic acid constructs of the
present invention contain one or more selectable marker genes to
provide a phenotypic trait for selection of transformed host cells.
Suitable marker genes include, but are not limited to those coding
for antimicrobial resistance such as, ampicillin (ampR), kanamycin,
chloramphenicol, tetracycline, streptomycin or spectinomycin (e.g.,
the aada gene); including but not limited to the streptomycin
phosphotransferase (spt) gene coding for streptomycin resistance,
the neomycin phosphotransferase (nptII) gene encoding kanamycin or
geneticin resistance, the nourseothricin acetyltransferase (nat1)
gene coding for nourseothricin resistance, the hygromycin
phosphotransferase (hpt) gene coding for hygromycin resistance,
genes encoding dihydrofolate reductase, phleomycin, or neomycin
resistance for eukaryotic cell culture, and tetracycline or
ampicillin resistance in E. coli, as well as other marker genes
that are well known in the art. Nucleic acid constructs of the
present invention typically comprise a fungal origin of
replication, such as, for example, a filamentous fungal or yeast
origin of replication. Typically, the recombinant nucleic acid
constructs of the present invention comprise a yeast origin of
replication. Examples include, but are not limited to constructs
containing autonomous replicating sequences, constructs containing
2 micron DNA including the autonomous replicating sequence and rep
genes, constructs containing centromeres like the CEN6, CEN4,
CEN11, CDN3 and autonomous replicating sequences, and other like
sequences that are well known in the art. Exemplary nucleic acid
constructs include constructs suitable for transforming yeast.
These include, but are not limited to episomal constructs based on
the yeast 2.mu. or CEN origin based plasmids like pYES2/CT,
pYES3/CT, pESC/His, pESC/Ura, pESC/Trp, pES/Leu, p427TEF, pRS405,
pRS406, pRS413, and other yeast-based constructs that are known in
the art.
In some embodiments, the nucleic acid constructs of the present
invention comprise elements to facilitate integration of the xylose
isomerase polynucleotide into a fungal host chromosome (i.e., the
genome), by either homologous or non-homologous recombination and
either site-directed or random mutagenesis. In some embodiments,
the nucleic acid constructs comprise elements that facilitate
homologous integration. In some embodiments, the xylose isomerase
polynucleotide is integrated at one or more site and is present in
one or more copies. In some embodiments, the nucleic acid construct
comprises the xylose isomerase polynucleotide and no promoter that
is operatively linked to the xylose isomerase polynucleotide. This
type of construct typically comprises genetic elements to
facilitate integration into the fungal host chromosome at a
location that is downstream of a native promoter (i.e., in the host
chromosome). In some embodiments, a second nucleic acid construct
is employed which comprises a promoter and genetic elements to
facilitate integration into the fungal host chromosome in a
location upstream of the targeted integration site of the xylose
isomerase polynucleotide. In some embodiments, the nucleic acid
construct comprises the xylose isomerase polynucleotide operatively
linked to a promoter or promoter and terminator sequences such that
all are integrated into the host chromosome (genome).
Genetic elements that facilitate integration by homologous
recombination are those having sequence homology to targeted
integration sites in the fungal host chromosome (genome). Suitable
sites that find use as targets for integration include, but are not
limited to the TY1 loci, the RDN loci, the ura3 locus, the GPD
locus, aldose reductase (GRE3) locus, etc. Those having ordinary
skill in the art will appreciate that additional sites for
integration can be readily identified using methods known in the
art, including but not limited to microarray analysis, metabolic
flux analysis, comparative genome hybridization analysis, etc.
Genetic elements or techniques which facilitate integration by
non-homologous recombination include, but are not limited to
restriction enzyme-mediated integration (REMI) (See e.g.,
Manivasakam et al., Mol. Cell Biol., 18(3):1736-1745 [1998], which
is incorporated herein by reference), transposon-mediated
integration, and other elements and methods that are well known in
the art.
In some embodiments, the nucleic acid constructs of the present
invention comprise at least one further recombinant polynucleotide
that is capable of conferring a desired phenotype to a fungal host
cell, particularly in the context of xylose fermentation. In some
embodiments, the recombinant polynucleotide that is capable of
conferring an improved phenotype to the fungal host cell is a
non-coding polynucleotide such as a regulatory polynucleotide, a
coding polynucleotide, or combination thereof.
Exemplary further desired phenotypes include, but are not limited
to increased transport of xylose into the host cell, increased
xylulose kinase activity, increased flux through the pentose
phosphate pathway, decreased sensitivity to catabolite repression,
increased tolerance to ethanol, increased tolerance to increased
osmolarity, increased tolerance to organic acids, reduced
production of by-products, and other similar properties related to
increasing flux through the pentose phosphate and glycolysis
pathways to produce a desired metabolic product/intermediate at
higher levels as compared to the corresponding wild-type host cell.
Typically, the desired metabolic product is an alcohol (e.g.,
ethanol).
In some embodiments, nucleic acid constructs comprising at least
one further polynucleotide that is capable of conferring a desired
phenotype to a fungal host cell comprise a polynucleotide encoding
a protein known to impact the desired phenotype, wherein the
polynucleotide is either native or heterologous to the fungal host
cell. In some embodiments, this polynucleotide is operatively
linked to its native promoter, or to a heterologous promoter (i.e.,
a promoter that is not associated with the polynucleotide in the
corresponding native gene). In some embodiments, the at least one
further polynucleotide is overexpressed. In some embodiments, the
nucleic acid constructs comprise multiple copies of a least one
polynucleotide. Suitable polynucleotides include, but are not
limited to those that facilitate overexpression of proteins known
to have an impact on the desired phenotype.
Exemplary recombinant polynucleotides that are capable of
conferring a desired phenotype to a fungal host cell include
recombinant polynucleotides (either wild-type or mutated forms)
which encode a xylose or hexose transporter, a xylulose kinase
(XKS), an enzyme from the pentose phosphate pathway (See e.g., FIG.
2A), a glycolytic enzyme (i.e., from the glycolytic metabolic
pathway; See e.g., FIG. 2B), and an ethanologenic enzyme (See e.g.,
FIG. 2C), regulatory sequences that enhance expression of these
sequences, and combinations thereof. Additional recombinant
polynucleotides (either wild-type or mutated forms) that find use
in the present invention include those that encode additional
proteins involved in the pentose phosphate, glycolysis, and
ethanologenic pathways (See e.g., FIGS. 2A-C).
Exemplary transporters include, but are not limited to GXF1, SUT1
and At6g59250 from Candida intermedia, Pichia stipitis and
Arabidopsis thaliana, respectively (See e.g., Runquist et al.,
Biotechnol. Biofuels, 3:5 [2010], which is incorporated herein by
reference), as well as HXT4, HXT5, HXT7, GAL2, AGT1, GXF2 (See
e.g., Matsushika et al., Appl. Microbiol. Biotechnol., 84:37-53
[2009], which is incorporated herein by reference). In some
embodiments, overexpression of native S. cerevisiae transporters is
desirable, particularly HXT5 and HXT7.
Particularly suitable recombinant polynucleotides include those
which encode: a xylulose kinase (XK); an enzyme from the pentose
phosphate pathway (e.g., a ribulose-5-phosphate 3-epimerase (RPE1),
a ribose-5-phosphate ketol-isomerase (RKI1), a transketolase
(TKL1), a transaldolase (TAL1), etc.); a glycolytic enzyme (e.g., a
hexokinase (HXK1/HXK2), a glyceraldehyde-3-phosphate dehydrogenase
(GAPDH), a pyruvate kinase (PVK2), etc.); and an ethanologenic
enzyme (e.g., a pyruvate decarboxylase, an alcohol dehydrogenase,
etc.).
Exemplary regulatory polynucleotides include promoters, enhancer,
terminator, and other regulatory elements that function to improve
the expression of polynucleotides in a fungal host cell,
particularly, a yeast host cell. These include, but are not limited
to the regulatory elements described hereinabove.
The nucleic acid constructs described herein are useful for
transforming fungal host cells to confer to these cells the
property of xylose utilization.
Recombinant Fungal Host Cells
The present invention provides a recombinant fungal host cell
comprising at least one xylose isomerase polynucleotide provided
herein. More specifically, the recombinant fungal host cell
comprises a polynucleotide sequence that encodes a polypeptide
which is capable of catalyzing the isomerization of D-xylose
directly to D-xylulose, wherein the polynucleotide is selected
from: (a) a polynucleotide that encodes a polypeptide comprising an
amino acid sequence that is at least about 70% identical to SEQ ID
NO:2; and (b) a polynucleotide that hybridizes under stringent
hybridization conditions to the complement of a polynucleotide
encoding a polypeptide having the amino acid sequence of SEQ ID
NO:2.
In some embodiments, the present invention provides a recombinant
fungal host cell comprising or transformed with a nucleic acid
construct of the present invention. In some embodiments, the xylose
isomerase polynucleotide is integrated into the host cell genome.
Typically, the recombinant fungal host cell is a filamentous fungal
or yeast host cell. More typically, the recombinant fungal host
cell is a yeast host cell.
The present invention also provides methods for producing a
recombinant fungal host cell, wherein the method comprises: (a)
providing a nucleic acid construct of the present invention,
wherein the nucleic acid construct comprises at least one xylose
isomerase polynucleotide provided herein; and (b) transforming a
fungal host cell with the nucleic acid construct to produce a
recombinant fungal host cell.
Introduction of the expression construct of the present invention
into the host cell can be accomplished using any suitable method,
including but not limited to calcium phosphate transfection,
DEAE-dextran mediated transfection, electroporation, or any other
suitable technique. Indeed, there are numerous methods known in the
art and described in various standard reference texts. In some
embodiments, the xylose isomerase polynucleotide sequence is
integrated into the host cell genome.
Suitable fungal host cells include yeast and filamentous fungal
host cells. In some embodiments, the fungal host cell is a yeast
cell. Exemplary yeast host cells that are useful in the practice of
the present invention include, but are not limited to Candida,
Hansenula, Saccharomyces, Schizosaccharomyces, Pichia,
Kluyveromyces, and Yarrowia. In some embodiments of the invention,
the yeast cell is Hansenula polymorpha, Saccharomyces cerevisiae,
Saccharomyces carlsbergensis, Saccharomyces diastaticus,
Saccharomyces norbensis, Saccharomyces kluyveri,
Schizosaccharomyces pombe, Pichia pastoris, Pichia finlandica,
Pichia trehalophila, Pichia kodamae, Pichia membranaefaciens,
Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia
quercuum, Pichia pijperi, Pichia stipitis, Pichia methanolica,
Pichia angusta, Kluyveromyces lactis, Candida albicans, or Yarrowia
lipolytica. In some embodiments, the yeast host cell is
Saccharomyces species. In some additional embodiments, the yeast
host cell is Saccharomyces cerevisiae.
Yeast strains that find use in the present invention include, but
are not limited to Lallemand 6469, Lallemand LYCC 6391, Lallemand
LYCC 6939, Lallemand LYCC 6469, Lallemand LYCC 6469 (all from
Lallemand, Inc., Montreal, Canada); NRRL YB-1952 (ARS (NRRL)
Collection, U.S. Department of Agriculture); and BY4741.
Suitable fungal host cells include, but are not limited to,
Ascomycota, Basidiomycota, Deuteromycota, Zygomycota, and Fungi
imperfecti. In some embodiments the fungal host cells are yeast
cells and filamentous fungal cells. The filamentous fungal host
cells of the present invention include all filamentous forms of the
subdivision Eumycotina and Oomycota. Filamentous fungi are
characterized by a vegetative mycelium with a cell wall composed of
chitin, cellulose and other complex polysaccharides. The
filamentous fungal host cells of the present invention are
morphologically distinct from yeast.
In some embodiments the filamentous fungal host cell may be a cell
of a species of, but not limited to Achlya, Acremonium,
Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis,
Cephalosporium, Chrysosporium, Cochliobolus, Corynascus,
Cryphonectria, Cryptococcus, Coprinus, Coriolus, Diplodia,
Endothia, Fusarium, Gibberella, Gliocladium, Humicola, Hypocrea,
Myceliophthora, Mucor, Neurospora, Penicillium, Podospora, Phlebia,
Piromyces, Pyricularia, Rhizomucor, Rhizopus, Schizophyllum,
Scytalidium, Sporotrichum, Talaromyces, Thermoascus, Thielavia,
Trametes, Tolypocladium, Trichoderma, Verticillium, Volvariella, or
teleomorphs, or anamorphs, and synonyms, basonyms, and/or taxonomic
equivalents thereof.
In some embodiments of the invention, the filamentous fungal host
cell is of the Aspergillus species, Ceriporiopsis species,
Chrysosporium species, Corynascus species, Fusarium species,
Humicola species, Neurospora species, Penicillium species,
Tolypocladium species, Tramates species, or Trichoderma
species.
Indeed, exemplary filamentous fungal host cells that find use in
the present invention include, but are not limited to a filamentous
fungal host cell of the Trichoderma species (e.g., T.
longibrachiatum, T. viride [e.g., ATCC 32098 and 32086], T. reesei
[NRRL 15709, ATTC 13631, 56764, 56765, 56466, 56767, and RL-P37 and
derivatives thereof; See e.g., Sheir-Neiss et al., Appl. Microbiol.
Biotechnol., 20:46-53 [1984], incorporated herein by reference), T.
koningii, and T. harzianum), as well as Hypocrea jecorina. The term
"Trichoderma" refers to any fungal strain that was previously
classified as Trichoderma or is currently classified as
Trichoderma.
In some embodiments of the present invention, the filamentous
fungal host cell is an Aspergillus species (e.g., A. awamori, A.
funigatus, A. japonicas, A. nidulans, A. niger. A. aculeatus, A.
foetidus, A. oryzae, A. sojae, or A. kawachi (See e.g., Kelly and
Hynes, EMBO J., 4:475479 [1985]; NRRL 3112, ATCC 11490, 22342,
44733, and 14331; Yelton et al., Proc. Natl. Acad. Sci. USA, 81,
1480-1474 [1984]; Tilburn et al., Gene 26, 205-221 [1982]; and
Johnston et al., EMBO J., 4:1307-1311 [1985], all of which are
incorporated herein by reference). In some embodiments of the
invention, the filamentous fungal host cell is a Fusarium species
(e.g., F. bacterioides, F. cerealis, F. crookwellense, F. culmorum,
F. graminaearum, F. graminum, F. oxysporum, F. rosium, or F.
venenatum). In some embodiments of the invention, the filamentous
fungal host cell is of a Neurospora species (e.g., N. crassa; See
e.g., Case, et al., Proc. Natl. Acad. Sci. USA, 76:5259-5263
[1979]; U.S. Pat. No. 4,486,553; and Kinsey and Rambosek, Mol.
Cell. Biol., 4:117-122 [1984], all of which are incorporated herein
by reference). In some embodiments of the invention, the
filamentous fungal host cell is of a Humicola species (e.g., H.
insolens. H. grisea, or H. lanuginose). In some embodiments of the
invention, the filamentous fungal host cell is a Mucor species
(e.g., M. miehei or M. circinelloides). In some embodiments of the
invention, the filamentous fungal host cell is a Rhizopus species
(e.g., R. oryzae or R. niveus). In some embodiments of the
invention, the filamentous fungal host cell is of a Penicillium
species (e.g., P. purpurogenum, P. chrysogenum, or P.
verruculosum). In some embodiments of the invention, the
filamentous fungal host cell is a Thielavia species (e.g., T.
terrestris). In some embodiments of the invention, the filamentous
fungal host cell is a Tolypocladium species (e.g., T. inflatum or
T. geodes). In some embodiments of the invention, the filamentous
fungal host cell is a Trametes species (e.g., T. villosa or T.
versicolor). In some embodiments of the invention, the filamentous
fungal host cell is a Chrysosporium specie, (e.g., C. lucknowense,
C. keratinophilum, C. tropicum, C. merdarium, C. inops, C.
pannicola, or C. zonatum). In some embodiments of the invention,
the filamentous fungal host cell is of the Myceliophthora species,
e.g., M. thermophila.
Strains that find use in the present invention include those that
are readily accessible to the public from a number of culture
collection, including but not limited to the American Type Culture
Collection (ATCC), Deutsche Sammlung von Mikroorganismen and
Zellkutlturen GmbH (DSM), Centraalbureau Voor Schimmelcultures
(CBS), and Agricultural Research Service Patent Culture Collection,
Northern Regional Research Center (NRRL).
Recombinant fungal host cells of the present invention are capable
of growth in a xylose-based culture medium (i.e., a culture medium
where xylose is the primary carbon source). In these xylose-based
culture media, the carbon source typically consists essentially of
xylose. In some xylose-based culture media, the carbon source
consists of xylose. Typically, the recombinant fungal host cell is
capable of faster growth in a xylose-based culture medium as
compared to the corresponding wild-type fungal host cell. In some
embodiments, the recombinant fungal host cell is capable of faster
growth in a xylose-based culture medium as compared to wild-type
Saccharomyces cerevisiae. Typically, the recombinant fungal host
cell is capable of growth at a rate of at least about 0.2 per hour
(h.sup.-1) in a xylose-based culture medium. More typically, the
growth rate is at least about 0.3 or 0.4 per hour (h.sup.-1).
Growth rate can be determined by optical density, cell counting
methods, and any other suitable method. Indeed, there are various
well known methods for determining cell growth that find use in the
present invention. In some embodiments, the recombinant fungal host
cell is capable of fermenting xylose at a rate of at least about 1
g/L/h in a xylose-based culture medium, and sometimes at a rate of
at least about 2 g/L/h in a xylose-based culture medium. Exemplary
xylose-based culture media include culture media which have been
formulated to contain xylose (See e.g., Example 2 herein), as well
as feedstock from a cellulosic saccharification process and/or
feedstock from a hemicellulose pre-treatment process (i.e., a
"hemicellulosic feedstock").
Recombinant fungal host cells of the present invention are also
capable of fermenting xylose when provided with a xylose based
culture medium. Typically, the recombinant fungal host cells
described herein are capable of fermenting xylose at a faster rate
compared to the corresponding wild-type fungal host cell. In some
embodiments, the recombinant fungal host cells are capable of
fermenting xylose at a rate of at least about 1 g/L/h and sometimes
at a rate of at least about 2 g/L/h. In some embodiments the
recombinant fungal host cells are capable of fermenting xylose at a
rate of at least 0.5 g/g CDW/h and sometimes at a rate of 0.25 g/g
CDW/h and other times at a rate of 0.1 g/g CDW/h. Exemplary
xylose-based culture media include culture media which have been
formulated to contain xylose, as well as feedstock from cellulosic
saccharification processes and/or feedstock from a hemicellulose
pre-treatment process (i.e., a "hemicellulosic feedstock").
In some embodiments, the fungal host cell is a wild-type fungal
cell, while in other embodiments, it is a mutated or otherwise
altered or engineered form of a wild-type fungal cell. Typically,
the fungal host cell (either wild-type or otherwise altered or
engineered) comprises polynucleotides encoding a xylulokinase and
one or more enzymes in the pentose phosphate, glycolytic, and/or
ethanologenic pathways. In some embodiments, the fungal host cell
comprises polynucleotides encoding a xylulokinase and all of the
enzymes in the pentose phosphate, glycolytic, and ethanologenic
pathways. In some embodiments, the fungal host cell comprises
recombinant polynucleotides encoding enzymes that are heterologous
to the fungal host cell (i.e., not native to the fungal host cell).
In some additional embodiments, the fungal host cell is engineered
to comprise other metabolic pathways that utilize
products/intermediates from the pentose phosphate, glycolytic,
and/or ethanologenic pathways to produce other desirable products.
For example, in some embodiments, the fungal host cell is
engineered to comprise a metabolic pathway for the biosynthesis of
a fatty alcohol or fatty acid (See e.g., WO 2007/136762, which is
incorporated herein by reference). In some embodiments, the fatty
alcohol or fatty acid is a C8-C20 fatty acid or fatty alcohol. In
some embodiments, the fungal host cell is altered or engineered to
overexpress any one or more of the polynucleotides encoding the
enzymes in one or more of these metabolic pathways.
In some embodiments, the recombinant fungal host cell of the
present invention further comprises genetic modifications in
addition to the xylose isomerase polynucleotide. In some
embodiments, in addition to having a xylose isomerase
polynucleotide described herein, the recombinant host cell
comprises at least one different recombinant polynucleotide that is
capable of conferring a further desired phenotype to the fungal
host cell. In some embodiments, the present invention provides a
recombinant fungal host cell comprising at least one Ruminococcus
flavefaciens xylose isomerase polynucleotide or variant thereof as
described herein, and at least one recombinant polynucleotide that
encodes a polypeptide which differs from the Ruminococcus
flavefaciens xylose isomerase or variant thereof, wherein the
recombinant polynucleotide imparts a desired phenotype to the
fungal host cell. It is contemplated that the recombinant
polynucleotide that is capable of conferring a desired phenotype to
the fungal host cell may be introduced to the fungal host cell on
the same nucleic construct as the xylose isomerase polynucleotide,
or on a separate nucleic acid construct. Nucleic acid constructs of
the present invention comprising both a xylose isomerase
polynucleotide and at least one further recombinant polynucleotide
capable of conferring a desired phenotype to the fungal host cell
are described above.
In some embodiments, the recombinant polynucleotide that is capable
of conferring a desired phenotype to the fungal host cell is a
non-coding polynucleotide (e.g., a regulatory polynucleotide, a
coding polynucleotide, or a combination thereof). As described
above, exemplary further desired phenotypes include, but are not
limited to increased transport of xylose into the host cell,
increased xylulose kinase activity, increased flux through the
pentose phosphate pathway, decreased sensitivity to catabolite
repression, increased tolerance to ethanol, increased tolerance to
increased osmolarity, increased tolerance to organic acids, reduced
production of by-products, and other like properties related to
increasing flux through the pentose phosphate, glycolysis, and/or
ethanologenic pathways to produce the desired metabolic
product/intermediate at higher levels as compared to the
corresponding wild-type host cell. In some embodiments, the desired
metabolic product is an alcohol (e.g., ethanol).
In some embodiments, recombinant fungal host cells comprising at
least one further polynucleotide capable of conferring a desired
phenotype to the fungal host cell comprise at least one
polynucleotide encoding a protein known to impact the desired
phenotype, wherein the polynucleotide is either native or
heterologous to the fungal host cell. In some embodiments, the
polynucleotide(s) are operatively linked to its native promoter,
while in other embodiments, the polynucleotide is operatively
linked to a heterologous promoter (i.e., one not associated with
the polynucleotide in the corresponding native gene). In some
embodiments, the polynucleotide is overexpressed. In some
embodiments, the recombinant fungal host cell comprises multiple
copies of the polynucleotide. Suitable polynucleotides include, but
are not limited to those that facilitate overexpression of proteins
known to have an impact on the desired phenotype. Therefore, in
some embodiments, the fungal host cell is altered or engineered to
overexpress one or more polynucleotides.
In some embodiments, recombinant polynucleotides that are capable
of imparting a desired phenotype to a fungal host cell include, but
are not limited to recombinant polynucleotides which encode a
xylose or hexose transporter, a xylulose kinase (XKS), an enzyme
from the pentose phosphate pathway (See e.g., FIG. 2A), a
glycolytic enzyme (i.e., from the metabolic pathway of glycolysis;
See e.g., FIG. 2B), and an ethanologenic enzyme (See e.g., FIG.
2C), the regulatory sequences associated with these sequences, and
any combination thereof.
Exemplary transporters that find use in the present invention
include, but are not limited to GXF1, SUT1 and At6g59250 from
Candida intermedia, Pichia stipitis, and Arabidopsis thaliana,
respectively (See e.g., Runquist et al., 84:37-53 [2010],
incorporated herein by reference), HXT4, HXT5, HXT7, GAL2, AGT1,
and GXF2, (See e.g., Matsushika et al., Appl. Microbiol.
Biotechnol., 84:37-53 [2009]). In some embodiments, overexpression
of native S. cerevisiae transporters is desirable, particularly
HXT5 and HXT7.
Particularly suitable recombinant polynucleotides include, but are
not limited to those that encode: a xylulose kinase (XK); an enzyme
from the pentose phosphate pathway (e.g., a ribulose-5-phosphate
3-epimerase (RPE1), a ribose-5-phosphate ketol-isomerase (RKI1), a
transketolase (TKL1), a transaldolase (TAL1), etc.); a glycolytic
enzyme (e.g., a hexokinase (HXK1/HXK2), a
glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a pyruvate kinase
(PVK2), etc.; and an ethanologenic enzyme (e.g., a pyruvate
decarboxylase, an alcohol dehydrogenase, etc.).
Exemplary regulatory polynucleotides include promoters, enhancer,
terminator, and other regulatory elements that function to improve
the expression of polynucleotides in a fungal host cell,
particularly, a yeast host cell, as described above.
In some embodiments, recombinant host cells of the present
invention comprise one or more native genes deleted from its
genome. In some embodiments, the deletion(s) cause removal or
diminishment of a biological activity that is otherwise exhibited
by the fungal host cell. In some embodiments, the cumulative effect
of the deletion(s) also leads to an improvement in a phenotype of
the fungal host cell. Any suitable method for deleting gene finds
use in the present invention. There are numerous methods well known
in the art. For example, in some embodiments, recombinant host
cells of the present invention have certain native genes deleted
from the host genome in order to improve the utilization of pentose
sugars (e.g., xylose), increase transport of xylose into the host
cell, increase xylulose kinase activity, increase flux through the
pentose phosphate pathway, decrease sensitivity to catabolite
repression, increase tolerance to ethanol/acetate, increase
tolerance to increased osmolarity, increase tolerance to organic
acids (low pH), reduce production of by-products, and other like
properties related to increasing flux through the relevant pathways
to produce ethanol and other desired metabolic products at higher
levels, where comparison is made with respect to the corresponding
host cell without the deletion(s). Genes targeted for deletion
include, but are not limited to genes encoding enzymes in the
pentose phosphate pathway, a glycolytic enzyme, and/or an
ethanologenic enzyme.
In some embodiments, other genes are targeted for deletion,
including but not limited to those encoding aldose reductase (GRE3)
(See e.g., Matsushika et al., Appl. Microbiol. Biotechnol.,
84:37-53 [2009]), sorbitol dehydrogenases (SOR1/SOR2), a glutamate
dehydrogenase (GDH1), a 6-phosphogluconate dehydrogenase (GND), a
glucose-5-phosphate dehydrogenase (ZWF1), and any enzyme for which
its deletion is known in the art to improve the utilization of a
pentose sugar, decrease by-product formation, and/or increase the
ethanol yield of a fungal host cell. The genes encoding these
enzymes in many fungi are known in the art. Those having ordinary
skill in the art appreciate that additional genes encoding these
enzymes can be readily identified by microarray analysis (See e.g.,
Sedlak et al., Yeast 21:671-684 [2004]), metabolic flux analysis
(See e.g Sonderegger et al., Appl. Environ. Microbiol.,
70(4):2307-2317 [2004]), in silico modeling (See e.g Hjersted et
al., Biotechnol. Bioengineer. 97(5):1190-1204 [2007]),
chemogenomics (See e.g Teixeira et al., Appl. Environ. Microbiol.,
75(18):5761-5772 [2009]), and other well known methods.
In some embodiments, the host cells employed in the practice of the
present invention are mutagenized and/or evolved to exhibit further
desired phenotypes, for example, further improvement in the
utilization of pentose sugars (e.g., xylose, arabinose, etc.),
increased transport of xylose into the host cell, increased
xylulose kinase activity, increased flux through the pentose
phosphate pathway, decreased sensitivity to catabolite repression,
increased tolerance to ethanol/acetate, increased tolerance to
increased osmolarity, increased tolerance to organic acids (low
pH), reduced production of by-products, and other like properties
related to increasing flux through the pentose phosphate and
glycolysis pathways to produce a desired metabolic
product/intermediate at higher levels. In some embodiments, the
desired metabolic product is an alcohol (e.g., ethanol). In some
embodiments, the host cells are mutagenized and/or evolved using
known methods either prior to or after transformation with the
xylose isomerase polynucleotide. These methods include, but are not
limited to classical mutagenesis, whole genome shuffling,
evolutionary engineering methods, which employ screening and/or
selection methods, or any combination of such well known
methods.
Classical mutagenesis methods include, but are not limited to
treatment of the host cell with a mutagen such as a chemical
mutagen or irradiation exposure (e.g., ultraviolet or
gamma-irradiation). Whole genome shuffling methods involving, for
example, recombination of genomic DNA between native genomic DNA
sequences and/or variants thereof, can be facilitated by sexual
mating, protoplast fusion methods and other methods well known in
the art (See e.g., WO 98/31837 and WO 2000/04190, incorporated
herein by reference). These methods are coupled with screening
and/or selection methods to identify altered fungal host cells that
exhibit the desired phenotype. For example, such methods find use
in altering or engineering a fungal host cell to overexpress one or
more desired polynucleotides.
Evolutionary engineering can be done by prolonged cultivation and
selection of strains under desired conditions through chemostat,
turbidostat or batch cultures. Evolutionary engineering methods can
be practiced under either aerobic or anaerobic conditions.
Selection strategies can be optimized by varying culture
conditions, for example, carbon source, nitrogen source, aeration,
pH and temperature. Methods for evolutionary engineering are well
known in the art (See e.g., Wisselink et al., Appl. Environ.
Microbiol., 75(4):907-914 [2009]; Kuyper et al., FEMS Yeast Res.,
5:399-409 [2005]; and Sauer, Adv. Biochem. Engineer. Biotechnol.,
73:129-169 [2001], all of which are incorporated herein by
reference).
Therefore, in some embodiments, the recombinant fungal host cell
comprising a xylose isomerase polynucleotide exhibits an improved
phenotype relative to the corresponding fungal host cell without
the xylose isomerase polynucleotide. In some embodiments, the
improved phenotype comprises further improvement in the utilization
of pentose sugars (e.g., xylose, arabinose, etc.), increased
transport of xylose into the host cell, increased xylulose kinase
activity, increased flux through the pentose phosphate pathway,
decreased sensitivity to catabolite repression, increased tolerance
to ethanol/acetate, increased tolerance to increased osmolarity,
increased tolerance to organic acids (low pH), and reduced
production of by products, or other properties.
Enzyme Mixtures
In some embodiments, the present invention provides an enzyme
mixture that comprises at least one xylose isomerase variant
polypeptide as provided herein. The enzyme mixture may be
cell-free, or in alternative embodiments, may not be separated from
host cells that secrete an enzyme mixture component. A cell-free
enzyme mixture typically comprises enzymes that have been separated
from cells. Cell-free enzyme mixtures can be prepared by any of a
variety of methodologies that are known in the art, such as
filtration or centrifugation methodologies. In some embodiments,
the enzyme mixtures are partially cell-free, substantially
cell-free, or entirely cell-free.
In some embodiments, at least one xylose isomerase variant and any
additional enzymes present in the enzyme mixture are secreted from
a single genetically modified fungal cell or by different microbes
in combined or separate fermentations. Similarly, in additional
embodiments, at least one xylose isomerase variant and any
additional enzymes present in the enzyme mixture are expressed
individually or in sub-groups from different strains of different
organisms and the enzymes are combined in vitro to make the enzyme
mixture. It is also contemplated that the xylose isomerases and any
additional enzymes in the enzyme mixture will be expressed
individually or in sub-groups from different strains of a single
organism, and the enzymes combined to make the enzyme mixture. In
some embodiments, all of the enzymes are expressed from a single
host organism, such as a genetically modified fungal cell.
In some embodiments, the enzyme mixture comprises at least one
cellulase, selected from cellobiohydrolase (CBH), endoglucanase
(EG), and/or beta-glucosidase (BG) cellulase. In some embodiments,
the cellobiohydrolase is T. reesei cellobiohydrolase II. In some
embodiments, the endoglucanase comprises a catalytic domain derived
from the catalytic domain of a Streptomyces avermitilis
endoglucanase. In some embodiments, at least one cellulase is
Acidothermus cellulolyticus, Thermobifida fusca, Humicola grisea or
a Chrysosporium sp. cellulose. Cellulase enzymes of the cellulase
mixture work together in decrystallizing and hydrolyzing the
cellulose from a biomass substrate to yield soluble sugars, such as
but not limited to glucose (See e.g., Brigham et al. in Wyman
([ed.], Handbook on Bioethanol, Taylor and Francis, Washington D.C.
[1995], pp 119-141, incorporated herein by reference).
Cellulase mixtures for efficient enzymatic hydrolysis of cellulose
are known (See e.g., Viikari et al., Adv. Biochem. Eng.
Biotechnol., 108:121-45 [2007]; and US Pat. Publns. 2009/0061484;
US 2008/0057541; and US 2009/0209009, each of which is incorporated
herein by reference). In some embodiments, mixtures of purified
naturally occurring or recombinant enzymes are combined with
cellulosic feedstock or a product of cellulose hydrolysis. In some
embodiments, one or more cell populations, each producing one or
more naturally occurring or recombinant cellulases, are combined
with cellulosic feedstock or a product of cellulose hydrolysis.
In some embodiments, at least one variant xylose isomerase
polypeptide of the present invention is present in mixtures
comprising enzymes other than cellulases that degrade cellulose,
hemicellulose, pectin, and/or lignocellulose.
A "hemicellulase" as used herein, refers to a polypeptide that can
catalyze hydrolysis of hemicellulose into small polysaccharides
such as oligosaccharides, or monomeric saccharides. Hemicellulloses
include xylan, glucuonoxylan, arabinoxylan, glucomannan and
xyloglucan. Hemicellulases include, for example, the following:
endoxylanases, b-xylosidases, a-L-arabinofuranosidases,
a-D-glucuronidases, feruloyl esterases, coumarolyl esterases,
a-galactosidases, b-galactosidases, b-mannanases, and
b-mannosidases. In some embodiments, the present invention provides
enzyme mixtures that comprise at least one xylose isomerase variant
of the present invention and one or more hemicellulases.
In some additional embodiments, the present invention provides at
least one xylose isomerase variant and at least one endoxylanase.
Endoxylanases (EC 3.2.1.8) catalyze the endohydrolysis of
1,4-.beta.-D-xylosidic linkages in xylans. This enzyme may also be
referred to as endo-1,4-.beta.-xylanase or 1,4-.beta.-D-xylan
xylanohydrolase. In some embodiments, an alternative is EC
3.2.1.136, a glucuronoarabinoxylan endoxylanase, an enzyme that is
able to hydrolyze 1,4 xylosidic linkages in
glucuronoarabinoxylans.
In some additional embodiments, the present invention provides at
least one xylose isomerase variant and at least one
.beta.-xylosidase. .beta.-xylosidases (EC 3.2.1.37) catalyze the
hydrolysis of 1,4-.beta.-D-xylans, to remove successive D-xylose
residues from the non-reducing termini. This enzyme may also be
referred to as xylan 1,4-.beta.-xylosidase, 1,4-.beta.-D-xylan
xylohydrolase, exo-1,4-.beta.-xylosidase or xylobiase.
In some additional embodiments, the present invention provides at
least one xylose isomerase variant and at least one
.alpha.-L-arabinofuranosidase. .alpha.-L-arabinofuranosidases (EC
3.2.1.55) catalyze the hydrolysis of terminal non-reducing
alpha-L-arabinofuranoside residues in alpha-L-arabinosides. The
enzyme acts on alpha-L-arabinofuranosides, alpha-L-arabinans
containing (1,3)- and/or (1,5)-linkages, arabinoxylans, and
arabinogalactans. Alpha-L-arabinofuranosidase is also known as
arabinosidase, alpha-arabinosidase, alpha-L-arabinosidase,
alpha-arabinofuranosidase, arabinofuranosidase, polysaccharide
alpha-L-arabinofuranosidase, alpha-L-arabinofuranoside hydrolase,
L-arabinosidase and alpha-L-arabinanase.
In some additional embodiments, the present invention provides at
least one xylose isomerase variant and at least one
alpha-glucuronidase. Alpha-glucuronidases (EC 3.2.1.139) catalyze
the hydrolysis of an alpha-D-glucuronoside to D-glucuronate and an
alcohol.
In some additional embodiments, the present invention provides at
least one xylose isomerase variant and at least one
acetylxylanesterase. Acetylxylanesterases (EC 3.1.1.72) catalyze
the hydrolysis of acetyl groups from polymeric xylan, acetylated
xylose, acetylated glucose, alpha-napthyl acetate, and
p-nitrophenyl acetate.
In some additional embodiments, the present invention provides at
least one xylose isomerase variant and at least one feruloyl
esterase. Feruloyl esterases (EC 3.1.1.73) have
4-hydroxy-3-methoxycinnamoyl-sugar hydrolase activity (EC 3.1.1.73)
that catalyzes the hydrolysis of the 4-hydroxy-3-methoxycinnamoyl
(feruloyl) group from an esterified sugar, which is usually
arabinose in "natural" substrates, to produce ferulate
(4-hydroxy-3-methoxycinnamate). Feruloyl esterase is also known as
ferulic acid esterase, hydroxycinnamoyl esterase, FAE-III,
cinnamoyl ester hydrolase, FAEA, cinnAE, FAE-I, or FAE-II.
In some additional embodiments, the present invention provides at
least one xylose isomerase variant and at least one coumaroyl
esterase. Coumaroyl esterases (EC 3.1.1.73) catalyze a reaction of
the form: coumaroyl-saccharide+H.sub.2O=coumarate+saccharide. In
some embodiments, the saccharide is an oligosaccharide or a
polysaccharide. This enzyme may also be referred to as
trans-4-coumaroyl esterase, trans-p-coumaroyl esterase, p-coumaroyl
esterase or p-coumaric acid esterase. The enzyme also falls within
EC 3.1.1.73 so may also be referred to as a feruloyl esterase.
In some additional embodiments, the present invention provides at
least one xylose isomerase variant and at least one
alpha-galactosidase. Alpha-galactosidases (EC 3.2.1.22) catalyze
the hydrolysis of terminal, non-reducing .alpha.-D-galactose
residues in .alpha.-D-galactosides, including galactose
oligosaccharides, galactomannans, galactans and arabinogalactans.
This enzyme may also be referred to as melibiase.
In some additional embodiments, the present invention provides at
least one xylose isomerase variant and at least one
beta-galactosidase. Beta-galactosidases (EC 3.2.1.23) catalyze the
hydrolysis of terminal non-reducing .beta.-D-galactose residues in
.beta.-D-galactosides. In some embodiments, the polypeptide is also
capable of hydrolyzing .alpha.-L-arabinosides. This enzyme may also
be referred to as exo-(1->4)-.beta.-D-galactanase or
lactase.
In some additional embodiments, the present invention provides at
least one xylose isomerase variant and at least one beta-mannanase.
Beta-mannanases (EC 3.2.1.78) catalyze the random hydrolysis of
1,4-.beta.-D-mannosidic linkages in mannans, galactomannans and
glucomannans. This enzyme may also be referred to as mannan
endo-1,4-.beta.-mannosidase or endo-1,4-mannanase.
In some additional embodiments, the present invention provides at
least one xylose isomerase variant and at least one
beta-mannosidase. Beta-mannosidases (EC 3.2.1.25) catalyze the
hydrolysis of terminal, non-reducing .beta.-D-mannose residues in
.beta.-D-mannosides. This enzyme may also be referred to as
mannanase or mannase.
In some embodiments one or more enzymes that degrade pectin are
included in enzyme mixtures that comprise at least one xylose
isomerase variant of the present invention. A pectinase catalyzes
the hydrolysis of pectin into smaller units such as oligosaccharide
or monomeric saccharides. In some embodiments, the enzyme mixtures
comprise any pectinase, for example an endo-polygalacturonase, a
pectin methyl esterase, an endo-galactanase, a pectin acetyl
esterase, an endo-pectin lyase, pectate lyase, alpha rhamnosidase,
an exo-galacturonase, an exo-polygalacturonate lyase, a
rhamnogalacturonan hydrolase, a rhamnogalacturonan lyase, a
rhamnogalacturonan acetyl esterase, a rhamnogalacturonan
galacturonohydrolase and/or a xylogalacturonase.
In some additional embodiments, the present invention provides at
least one xylose isomerase variant and at least one
endo-polygalacturonase. Endo-polygalacturonases (EC 3.2.1.15)
catalyze the random hydrolysis of 1,4-.alpha.-D-galactosiduronic
linkages in pectate and other galacturonans. This enzyme may also
be referred to as polygalacturonase pectin depolymerase, pectinase,
endopolygalacturonase, pectolase, pectin hydrolase, pectin
polygalacturonase, poly-.alpha.-1,4-galacturonide glycanohydrolase,
endogalacturonase; endo-D-galacturonase or
poly(1,4-.alpha.-D-galacturonide) glycanohydrolase.
In some additional embodiments, the present invention provides at
least one xylose isomerase variant and at least one pectin methyl
esterase. Pectin methyl esterases (EC 3.1.1.11) catalyze the
reaction: pectin+n H2O=n methanol+pectate. The enzyme may also been
known as pectinesterase, pectin demethoxylase, pectin methoxylase,
pectin methylesterase, pectase, pectinoesterase or pectin
pectylhydrolase.
In some additional embodiments, the present invention provides at
least one xylose isomerase variant and at least one
endo-galactanase. Endo-galactanases (EC 3.2.1.89) catalyze the
endohydrolysis of 1,4-.beta.-D-galactosidic linkages in
arabinogalactans. The enzyme may also be known as arabinogalactan
endo-1,4-.beta.-galactosidase, endo-1,4-.beta.-galactanase,
galactanase, arabinogalactanase or arabinogalactan
4-.beta.-D-galactanohydrolase.
In some additional embodiments, the present invention provides at
least one xylose isomerase variant and at least one pectin acetyl
esterase. Pectin acetyl esterases catalyze the deacetylation of the
acetyl groups at the hydroxyl groups of GaIUA residues of
pectin.
In some additional embodiments, the present invention provides at
least one xylose isomerase variant and at least one endo-pectin
lyase. Endo-pectin lyases (EC 4.2.2.10) catalyze the eliminative
cleavage of (1.fwdarw.4)-.alpha.-D-galacturonan methyl ester to
give oligosaccharides with
4-deoxy-6-O-methyl-.alpha.-D-galact-4-enuronosyl groups at their
non-reducing ends. The enzyme may also be known as pectin lyase,
pectin trans-eliminase; endo-pectin lyase, polymethylgalacturonic
transeliminase, pectin methyltranseliminase, pectolyase, PL, PNL or
PMGL or (1.fwdarw.4)-6-O-methyl-.alpha.-D-galacturonan lyase.
In some additional embodiments, the present invention provides at
least one xylose isomerase variant and at least one pectate lyase.
Pectate lyases (EC 4.2.2.2) catalyze the eliminative cleavage of
(1.fwdarw.4)-.alpha.-D-galacturonan to give oligosaccharides with
4-deoxy-.alpha.-D-galact-4-enuronosyl groups at their non-reducing
ends. The enzyme may also be known polygalacturonic transeliminase,
pectic acid transeliminase, polygalacturonate lyase, endopectin
methyltranseliminase, pectate transeliminase, endogalacturonate
transeliminase, pectic acid lyase, pectic lyase,
.alpha.-1,4-D-endopolygalacturonic acid lyase, PGA lyase, PPase-N,
endo-.alpha.-1,4-polygalacturonic acid lyase, polygalacturonic acid
lyase, pectin trans-eliminase, polygalacturonic acid
trans-eliminase or (1.fwdarw.4)-.alpha.-D-galacturonan lyase.
In some additional embodiments, the present invention provides at
least one xylose isomerase variant and at least one
alpha-rhamnosidase. Alpha-rhamnosidases (EC 3.2.1.40) catalyze the
hydrolysis of terminal non-reducing .alpha.-L-rhamnose residues in
.alpha.-L-rhamnosides or alternatively in rhamnogalacturonan. This
enzyme may also be known as .alpha.-L-rhamnosidase T,
.alpha.-L-rhamnosidase N or .alpha.-L-rhamnoside
rhamnohydrolase.
In some additional embodiments, the present invention provides at
least one xylose isomerase variant and at least one
exo-galacturonase. Exo-galacturonases (EC 3.2.1.82) hydrolyze
pectic acid from the non-reducing end, releasing digalacturonate.
The enzyme may also be known as exo-poly-.alpha.-galacturonosidase,
exopolygalacturonosidase or exopolygalacturanosidase.
In some additional embodiments, the present invention provides at
least one xylose isomerase variant and at least one
exo-galacturonase. Exo-galacturonases (EC 3.2.1.67) catalyze a
reaction of the following type:
(1,4-.alpha.-D-galacturonide)n+H2O=(1,4-.alpha.-D-galacturonide)n-i-
+D-galacturonate. The enzyme may also be known as galacturan
1,4-.alpha.-galacturonidase, exopolygalacturonase,
poly(galacturonate) hydrolase, exo-D-galacturonase,
exo-D-galacturonanase, exopoly-D-galacturonase or
poly(1,4-.alpha.-D-galacturonide) galacturonohydrolase.
In some additional embodiments, the present invention provides at
least one xylose isomerase variant and at least one
exopolygalacturonate lyase. Exopolygalacturonate lyases (EC
4.2.2.9) catalyze eliminative cleavage of
4-(4-deoxy-.alpha.-D-galact-4-enuronosyl)-D-galacturonate from the
reducing end of pectate (i.e. de-esterified pectin). This enzyme
may be known as pectate disaccharide-lyase, pectate exo-lyase,
exopectic acid transeliminase, exopectate lyase,
exopolygalacturonic acid-trans-eliminase, PATE, exo-PATE, exo-PGL
or (1.fwdarw.4)-.alpha.-D-galacturonan
reducing-end-disaccharide-lyase.
In some additional embodiments, the present invention provides at
least one xylose isomerase variant and at least one
rhamnogalacturonanase. Rhamnogalacturonanases hydrolyze the linkage
between galactosyluronic acid and rhamnopyranosyl in an
endo-fashion in strictly alternating rhamnogalacturonan structures,
consisting of the disaccharide
[(1,2-alpha-L-rhamnoyl-(1,4)-alpha-galactosyluronic acid].
In some additional embodiments, the present invention provides at
least one xylose isomerase variant and at least one
rhamnogalacturonan lyase Rhamnogalacturonan lyases cleave
.alpha.-L-Rhap-(1.fwdarw.4)-.alpha.-D-GalpA linkages in an
endo-fashion in rhamnogalacturonan by beta-elimination.
In some additional embodiments, the present invention provides at
least one xylose isomerase variant and at least one
rhamnogalacturonan acetyl esterase Rhamnogalacturonan acetyl
esterases catalyze the deacetylation of the backbone of alternating
rhamnose and galacturonic acid residues in rhamnogalacturonan.
In some additional embodiments, the present invention provides at
least one xylose isomerase variant and at least one
rhamnogalacturonan galacturonohydrolase Rhamnogalacturonan
galacturonohydrolases hydrolyze galacturonic acid from the
non-reducing end of strictly alternating rhamnogalacturonan
structures in an exo-fashion. This enzyme may also be known as
xylogalacturonan hydrolase.
In some additional embodiments, the present invention provides at
least one xylose isomerase variant and at least one endo-arabinase.
Endo-arabinanases (EC 3.2.1.99) catalyze endohydrolysis of
1,5-.alpha.-arabinofuranosidic linkages in 1,5-arabinans. The
enzyme may also be known as endo-arabinase, arabinan
endo-1,5-.alpha.-L-arabinosidase, endo-1,5-.alpha.-L-arabinanase,
endo-.alpha.-1,5-arabanase; endo-arabanase or
1,5-.alpha.-L-arabinan 1,5-.alpha.-L-arabinanohydrolase.
In some additional embodiments, the present invention provides at
least one xylose isomerase variant and at least one enzyme that
participates in lignin degradation in an enzyme mixture. Enzymatic
lignin depolymerization can be accomplished by lignin peroxidases,
manganese peroxidases, laccases and cellobiose dehydrogenases
(CDH), often working in synergy. These extracellular enzymes are
often referred to as "lignin-modifying enzymes" or "LMEs." Three of
these enzymes comprise two glycosylated heme-containing
peroxidases: lignin peroxidase (LIP); Mn-dependent peroxidase
(MNP); and, a copper-containing phenoloxidase laccase (LCC).
In some additional embodiments, the present invention provides at
least one xylose isomerase variant and at least one laccase.
Laccases are copper containing oxidase enzymes that are found in
many plants, fungi and microorganisms. Laccases are enzymatically
active on phenols and similar molecules and perform a one electron
oxidation. Laccases can be polymeric and the enzymatically active
form can be a dimer or trimer.
In some additional embodiments, the present invention provides at
least one xylose isomerase variant and at least one Mn-dependent
peroxidase. The enzymatic activity of Mn-dependent peroxidase (MnP)
in is dependent on Mn2+. Without being bound by theory, it has been
suggested that the main role of this enzyme is to oxidize Mn2+ to
Mn3+ (See e.g, Glenn et al., Arch. Biochem. Biophys., 251:688-696
[1986]). Subsequently, phenolic substrates are oxidized by the Mn3+
generated.
In some additional embodiments, the present invention provides at
least one xylose isomerase variant and at least one lignin
peroxidase. Lignin peroxidase is an extracellular heme that
catalyses the oxidative depolymerization of dilute solutions of
polymeric lignin in vitro. Some of the substrates of LiP, most
notably 3,4-dimethoxybenzyl alcohol (veratryl alcohol, VA), are
active redox compounds that have been shown to act as redox
mediators. VA is a secondary metabolite produced at the same time
as LiP by ligninolytic cultures of P. chrysosporium and without
being bound by theory, has been proposed to function as a
physiological redox mediator in the LiP-catalyzed oxidation of
lignin in vivo (See e.g., Harvey, et al., FEBS Lett., 195:242-246
[1986]).
In some additional embodiments, the present invention provides at
least one xylose isomerase variant and at least one protease and/or
a lipase that participates in cellulose degradation.
As used herein, "protease" includes enzymes that hydrolyze peptide
bonds (peptidases), as well as enzymes that hydrolyze bonds between
peptides and other moieties, such as sugars (glycopeptidases). Many
proteases are characterized under EC 3.4, and are suitable for use
in the present invention. Some specific types of proteases include,
cysteine proteases including pepsin, papain and serine proteases
including chymotrypsins, carboxypeptidases and
metalloendopeptidases.
As used herein, "lipase" includes enzymes that hydrolyze lipids,
fatty acids, and acylglycerides, including phosphoglycerides,
lipoproteins, diacylglycerols, and the like. In plants, lipids are
used as structural components to limit water loss and pathogen
infection. These lipids include waxes derived from fatty acids, as
well as cutin and suberin.
In some additional embodiments, the present invention provides at
least one xylose isomerase variant and at least one expansin or
expansin-like protein, such as a swollenin (See e.g., Salheimo et
al., Eur. J. Biochem., 269:4202-4211 [2002]) or a swollenin-like
protein. Expansins are implicated in loosening of the cell wall
structure during plant cell growth. Expansins have been proposed to
disrupt hydrogen bonding between cellulose and other cell wall
polysaccharides without having hydrolytic activity. In this way,
they are thought to allow the sliding of cellulose fibers and
enlargement of the cell wall. Swollenin, an expansin-like protein
contains an N-terminal Carbohydrate Binding Module Family 1 domain
(CBD) and a C-terminal expansin-like domain. In some embodiments,
an expansin-like protein or swollenin-like protein comprises one or
both of such domains and/or disrupts the structure of cell walls
(such as disrupting cellulose structure), optionally without
producing detectable amounts of reducing sugars.
In some additional embodiments, the present invention provides at
least one xylose isomerase variant and at least one polypeptide
product of a cellulose integrating protein, scaffoldin or a
scaffoldin-like protein, for example CipA or CipC from Clostridium
thermocellum or Clostridium cellulolyticum respectively.
Scaffoldins and cellulose integrating proteins are multi-functional
integrating subunits which may organize cellulolytic subunits into
a multi-enzyme complex. This is accomplished by the interaction of
two complementary classes of domain (i.e. a cohesion domain on
scaffoldin and a dockerin domain on each enzymatic unit). The
scaffoldin subunit also bears a cellulose-binding module that
mediates attachment of the cellulosome to its substrate. A
scaffoldin or cellulose integrating protein for the purposes of
this invention may comprise one or both of such domains.
In some additional embodiments, the present invention provides at
least one xylose isomerase variant and at least one cellulose
induced protein or modulating protein, for example as encoded by
cip1 or cip2 gene or similar genes from Trichoderma reesei (See
e.g., Foreman et al., J. Biol. Chem., 278:31988-31997 [2003]).
In some additional embodiments, the present invention provides at
least one xylose isomerase variant and at least one member of each
of the classes of the polypeptides described above, several members
of one polypeptide class, or any combination of these polypeptide
classes to provide enzyme mixtures suitable for various uses.
Other Components of Xylose Isomerase Compositions
In some embodiments, xylose isomerase polypeptides of the present
invention are used in combination with other optional ingredients
such as at least one buffer, surfactant, and/or scouring agent. In
some embodiments at least one buffer is used with at least one
xylose isomerase polypeptide of the present invention (optionally
combined with other enzymes) to maintain a desired pH within the
solution in which the xylose isomerase is employed. The exact
concentration of buffer employed will depend on several factors
which the skilled artisan can determine. Suitable buffers are well
known in the art. In some embodiments, at least one surfactant is
used in with at least one xylose isomerase of the present
invention. Suitable surfactants include any surfactant compatible
with the xylose isomerase(s) and, optionally, with any other
enzymes being used in the mixture. Exemplary surfactants include an
anionic, a non-ionic, and ampholytic surfactants. Suitable anionic
surfactants include, but are not limited to, linear or branched
alkylbenzenesulfonates; alkyl or alkenyl ether sulfates having
linear or branched alkyl groups or alkenyl groups; alkyl or alkenyl
sulfates; olefinsulfonates; alkanesulfonates, and the like.
Suitable counter ions for anionic surfactants include, for example,
alkali metal ions, such as sodium and potassium; alkaline earth
metal ions, such as calcium and magnesium; ammonium ion; and
alkanolamines having from 1 to 3 alkanol groups of carbon number 2
or 3. Ampholytic surfactants suitable for use in the practice of
the present invention include, for example, quaternary ammonium
salt sulfonates, betaine-type ampholytic surfactants, and the like.
Suitable nonionic surfactants generally include polyoxalkylene
ethers, as well as higher fatty acid alkanolamides or alkylene
oxide adduct thereof, fatty acid glycerine monoesters, and the
like. Mixtures of surfactants also find use in the present
invention, as is known in the art.
Fermentation
The present invention provides processes for producing fermentation
products, wherein the method comprises: (a) providing the
recombinant fungal cell of the present invention; (b) providing a
fermentation medium comprising xylose; (c) contacting the
fermentation medium with the recombinant fungal cell under
conditions suitable for generating the fermentation product; and
optionally (d) recovering the fermentation product. In some
embodiments, the fermentation product is an alcohol (e.g., ethanol,
butanol, etc.), a fatty alcohol (e.g., a C8-C20 fatty alcohol), a
fatty acid (e.g., a C8-C20 fatty acid), lactic acid,
3-hydroxypropionic acid, acrylic acid, acetic acid, succinic acid,
citric acid, malic acid, fumaric acid, an amino acid,
1,3-propanediol, ethylene, glycerol, and/or a .beta.-lactam (e.g.,
cephalosporin). However, it is contemplated that other fermentation
products will be produced using the methods of the present
invention.
In some embodiments, the fermentation medium is feedstock from a
cellulosic saccharification process and/or feedstock from a
hemicellulose pre-treatment process. Such feedstocks include, but
are not limited to carbohydrates (e.g., lignocellulose, xylans,
cellulose, starch, etc.), other sugars (e.g., glucose, xylose,
arabinose, etc.), and other compositions. Compositions of
fermentation media suitable for the growth of yeast and filamentous
fungi are well known in the art and there are various reference
texts that provide recipes for these media. Fermentation conditions
suitable for generating desired fermentation products are well
known in the art and any suitable method finds use in the present
invention. In some embodiments, the fermentation process is carried
out under aerobic or microaerophilic (i.e., where the concentration
of oxygen is less than that in air), or anaerobic conditions. In
some embodiments, fermentation is conducted under anaerobic
conditions (i.e., no detectable oxygen), or less than about 5,
about 2.5, or about 1 mmol/L/h oxygen. In the absence of oxygen,
the NADH produced in glycolysis cannot be oxidized by oxidative
phosphorylation. Under anaerobic conditions, pyruvate or a
derivative thereof may be utilized by the host cell as an electron
and hydrogen acceptor in order to generated NAD+. In some
embodiments of the present invention, when the fermentation process
is carried out under anaerobic conditions, pyruvate may be reduced
to a fermentation product such as ethanol, butanol, lactic acid,
3-hydroxypropionic acid, acrylic acid, acetic acid, succinic acid,
citric acid, malic acid, fumaric acid, an amino acid,
1,3-propanediol, ethylene, glycerol, and/or a .beta.-lactam (e.g.,
a cephalosporin).
The fermentation process is typically run at a temperature that is
optimal for the recombinant fungal cell. For example, in some
embodiments, the fermentation process is performed at a temperature
in the range of from about 25.degree. C. to about 42.degree. C.
Typically the process is carried out a temperature that is less
than about 38.degree. C., less than about 35.degree. C., less than
about 33.degree. C., or less than about 38.degree. C., but at least
about 20.degree. C., 22.degree. C., or 25.degree. C.
In some embodiments, recombinant host cells of the present
invention are grown under batch or continuous fermentation
conditions. Classical batch fermentation is a closed system,
wherein the composition of the medium is set at the beginning of
the fermentation and is not subject to artificial alterations
during the fermentation. A variation of the batch system is a
fed-batch fermentation, which also finds use in the present
invention. In this variation, the substrate is added in increments
as the fermentation progresses. Fed-batch systems are useful when
catabolite repression is likely to inhibit the metabolism of the
cells and/or where it is desirable to have limited amounts of
substrate in the medium. Batch and fed-batch fermentations are
common and well known in the art. Continuous fermentation is an
open system where a defined fermentation generally maintains the
culture at a constant high density where cells are primarily in log
phase growth. Continuous fermentation systems strive to maintain
steady state growth conditions. Methods for modulating nutrients
and growth factors for continuous fermentation processes, as well
as techniques for modulating nutrients and growth factors for
continuous fermentation processes as well as techniques for
maximizing the rate of product formation are well known in the art
of industrial microbiology.
The foregoing and other aspects of the invention may be better
understood in connection with the following non-limiting
examples.
EXPERIMENTAL
The present invention is described in further detail in the
following Examples, which are not in any way intended to limit the
scope of the invention as claimed.
In the experimental disclosure below, the following abbreviations
apply: ppm (parts per million); M (molar); mM (millimolar), uM and
.mu.M (micromolar); nM (nanomolar); mol (moles); gm and g (gram);
mg (milligrams); ug and .mu.g (micrograms); L and l (liter); ml and
mL (milliliter); cm (centimeters); mm (millimeters); um and .mu.m
(micrometers); sec. (seconds); min(s) (minute(s)); h(s) and hr(s)
(hour(s)); U (units); MW (molecular weight); rpm (rotations per
minute); .degree. C. (degrees Centigrade); DNA (deoxyribonucleic
acid); RNA (ribonucleic acid); CDW (cell dry weight); HPLC (high
pressure liquid chromatography); HMF (hydroxymethylfurfural); YPD
(yeast extract 10 g/L; peptone 20 g/L; dextrose 20 g/L);
propagation medium (160 g/l glucose, 40 g/l xylose, 4.5 g/l
arabinose, 20 g/l yeast extract, 6 g/l acetic acid, 0.6 g/l
furfural, 0.9 g/l hydroxymethylfurfural with a vitamin solution
added to final concentrations of 0.05 mg/l biotin, 1 mg/l calcium
pantothenate, 1 mg/l nicotinic acid, 1 mg/l myoinositol, 1 mg/l
thiamine chloride hydrochloride, 1 mg/l pyridoxal hydrochloride
potassium iodide and a trace element solution added to final
concentrations of 0.403 .mu.M EDTA, 15.6 .mu.M ZnSO4, 5 .mu.M
MnCl2, 1.3 .mu.M CoCl2, 1.2 .mu.M CuSO4, 1.6 .mu.M disodium
molybdate, 30.6 .mu.M CaCl2, 10.8 .mu.M FeSO4, 16.2 .mu.M boric
acid, 0.6 .mu.M potassium iodide, 5 g/l NH4SO4, 3 g/l K2PO4, 0.5
g/l MgSO4 and pH adjusted to 5.0 with NaOH); ARS (ARS Culture
Collection or NRRL Culture Collection, Peoria, Ill.); Lallemand
(Lallemand Ethanol Technology, Milwaukee, Wis.); Dualsystems
(Dualsystems Biotech AG, Basel, Switzerland); Megazyme (Megazyme
International Ireland, Ltd., Wicklow, Ireland); Dasgip (Dasgip
Biotools, LLC, Shrewsbury, Mass.); Difco (Difco Laboratories, BD
Diagnostic Systems, Detroit, Mich.); PCRdiagnostics
(PCRdiagnostics, E coli SRO, Slovak Republic); Agilent (Agilent
Technologies, Inc., Santa Clara, Calif.); and Bio-Rad (Bio-Rad
Laboratories, Hercules, Calif.).
Example 1
Vector Construction
A polynucleotide encoding the xylose isomerase from Ruminococcus
flavefaciens was synthesized with codons optimized for expression
in yeast with the following 5' and 3' flanks:
TABLE-US-00004 (SEQ ID NO: 5) 5'-GGATCCCAAACAAA (SEQ ID NO: 6)
3'-TAACATATG
These flanks were used to introduce 5'-BamH1 and 3'-Nde1
restriction sites flanking the gene. The codon-optimized
polynucleotide encoding the Ruminococcus flavefaciens xylose
isomerase corresponds to SEQ ID NO:3, which is provided below.
TABLE-US-00005 (SEQ ID NO: 3)
ATGGAATTTTTCTCCAACATCGGAAAAATCCAATACCAAGGTCCAAAATC
CACAGATCCTTTGTCTTTTAAATATTATAATCCTGAAGAAGTAATCAACG
GTAAGACCATGAGGGAGCATTTGAAATTCGCTCTATCCTGGTGGCACACT
ATGGGTGGCGATGGTACTGATATGTTCGGATGTGGTACTACGGACAAGAC
CTGGGGTCAATCCGACCCAGCGGCAAGAGCTAAGGCCAAAGTTGATGCTG
CTTTCGAAATTATGGATAAGCTGAGCATTGATTACTACTGCTTCCATGAT
AGAGACCTTTCTCCAGAATATGGCTCCTTGAAAGCGACCAATGATCAACT
GGACATTGTTACTGATTACATCAAGGAGAAGCAGGGCGATAAATTCAAGT
GTTTATGGGGCACTGCTAAATGCTTTGATCACCCCAGGTTCATGCACGGT
GCAGGAACTTCTCCTAGTGCCGATGTTTTCGCTTTTTCTGCTGCGCAAAT
AAAGAAAGCATTAGAATCTACCGTCAAGTTGGGCGGTAATGGTTATGTCT
TTTGGGGTGGTAGAGAAGGTTACGAGACCCTGCTGAATACTAACATGGGC
TTAGAACTGGACAACATGGCTAGGCTAATGAAGATGGCCGTAGAATACGG
TAGGTCTATTGGATTCAAAGGTGACTTCTACATCGAGCCTAAACCCAAGG
AACCTACTAAGCACCAGTACGACTTCGACACTGCTACCGTATTAGGTTTT
TTAAGGAAGTACGGGTTGGATAAAGACTTCAAGATGAACATCGAAGCCAA
TCACGCCACACTAGCACAACACACATTCCAGCATGAGTTACGTGTGGCTA
GGGATAACGGTGTATTCGGTTCTATTGATGCTAACCAAGGTGACGTATTG
TTAGGATGGGACACGGATCAATTCCCCACAAACATTTATGATACTACTAT
GTGTATGTATGAGGTCATTAAAGCCGGTGGTTTCACAAATGGCGGCCTGA
ACTTTGATGCGAAAGCTCGTAGGGGTTCATTCACGCCTGAAGATATTTTC
TATAGTTACATTGCTGGTATGGATGCTTTCGCGTTAGGGTTTAGAGCAGC
TCTTAAATTGATTGAAGACGGTAGAATTGACAAGTTTGTGGCTGACAGGT
ATGCCTCTTGGAATACCGGTATTGGTGCAGATATTATTGCCGGAAAAGCC
GATTTTGCATCATTGGAAAAATATGCTTTGGAAAAAGGTGAAGTTACCGC
GTCATTGTCTTCTGGTAGACAAGAGATGCTGGAATCTATTGTCAACAACG
TATTGTTTAGTTTGTAATAA
The yeast vector p427TEF (Dualsystems) was used for gene
expression. This vector contains a kanamycin resistance gene that
allows for selection in yeast, an ampicillin resistance gene that
allows for selection in E. coli, and a 2 micron origin of
replication that allows for propagation of plasmids in high copy
numbers in yeast. For cloning the xylose isomerase gene, p427TEF
was digested with SacI and XhoI restriction enzymes. The larger
fragment (6235 bp) was ligated with an oligomer of the following
sequence:
TABLE-US-00006 (SEQ ID NO: 7)
5'GAGCTCACGGATCCGTCATATGCTAGATCTCTGAATTCTTACTAGTTC
GACGTCTACCTAGGCAGTCGACACGCGGCCGCTTCTCGAG 3'
to introduce a new multiple cloning site (MCS) with desired
restriction sites. Using the new MCS, the TEF1 promoter of S.
cerevisiae was re-introduced in the vector using SacI/BamHI
restriction sites resulting in vector PLS1567. The codon-optimized
xylose isomerase gene was cloned in PLS1567 downstream of the TEF1
promoter using BamHI/NdeI restriction sites. The expression
construct, PLS4420 is depicted in FIG. 6.
Plasmids containing polynucleotides encoding xylose isomerase
homologues of the R. flavefaciens xylose isomerase from
Epulopiscium sp. 'N.t. morphotype B' (PLS4418; SEQ ID NO:8
(polynucleotide sequence), SEQ ID NO:9 (encoded amino acid
sequence), Alkaliphilus metalliredigens QYMF (PLS4416; SEQ ID NO:10
(polynucleotide sequence), SEQ ID NO:11 (encoded amino acid
sequence), Fusobacterium mortiferum ATCC 9817 (PLSX4417; SEQ ID
NO:12; polynucleotide sequence), SEQ ID NO:13 (encoded amino acid
sequence), Clostridium cellulolyticum H10 (PLS4419; SEQ ID NO:14;
polynucleotide sequence), SEQ ID NO:15 (encoded amino acid
sequence) and Clostridium phytofermentans (PLS1569; SEQ ID NO:16;
polynucleotide sequence), and SEQ ID NO:17 (encoded amino acid
sequence) were also individually cloned into vector PLS1567. The
native polynucleotide sequences from these microorganisms were
codon optimized for yeast. These sequences (SEQ ID NOS:8-17) are
provided below.
TABLE-US-00007 (SEQ ID NO: 8)
ATGGTGAACGGTTTGACCAACATCCCACCAGTCAAATTCGAAGGTAGAGA
CTCCAAAAAAGCATTGTCTTTTAAATATTATAATCCTGATGAAATGATCC
AAGGTAAGAAAATGAAGGATTATTTGAAATTCGCTATGTCCTATTGGCAC
ACTTTGTGTGGCGATGGTACTGATCCATTCGGATCATCTACTATTGACAG
GGACTACAGTGGCCAAACCCCAATGGAAAAAGCTAAGACCAAAGCTGATG
TTGCTTTCGCACTGATGCAAATTCTGGGCATCGAGTACTTCTGCTTCCAT
GATTTGGACATTGCTCCAACAGGTAACTCCTTGAAAGAGTTGAAGAATAA
TCTGATCGAGATTACTGATTACATCAAGGGGTTGATGGACAAAACCGGCA
TCAAGTTGTTATGGGGCACTGCTAACTGCTTTAGTCACCCCAGGTACATG
AACGGTGCAGGAACTTCTCCTCAAGCCGATATTTTCGCTTGTGCTGCTGC
GCAAATAAAGAACGCTATAGATGCGACCATCAAGTTGGGCGGTACGGGTT
ATGTCTTTTGGGGTGGTAGAGAAGGTTACGAGACCCTGAATACTAACATG
GAAATAGAACTGGACAACATGGCTAAGCTAATGCACATGGCCGTAGATTA
CGCTAGGTCTAAAGGATTCACCGGTGACTTCTACATCGAGCCTAAACCCA
AGGAACCTACTAAGCACCAGTACGACTTCGACGTTGCTACCGTAGTAGGT
TTTTTAAGGAAGTACGGGTTGGATAAAGACTTCAAGATGAACATCGAAGC
CAATCACGCCACACTAGCAGGCCACACATTCCAGCATGAGTTAAATGTGG
CTAGGGTAAACAATGTATTCGGTTCTATTGATGCTAACCAAGGTGACCTA
TTGTTAGGATGGGACACGGATCAATTCCCCACAAACGTTTATGATACTAC
TCTTTGTATGCTGGAGGTCATTAAAGCCGGTGGTTTCACAAATGGCGGCC
TGAACTTTGATGCGAAAGTTCGTAGGGCTTCATACACGATGGAAGATATT
ATCTTGGCTTACATTTCTGGTATGGATACTTTCGCGTTAGGGTTAAAAAT
AGCTAATAAAATCATTGAAGACGGTAGAATTGACGAGTTTGTGTCTAGGA
GGTATGCCTCTTACAAGACCGGTATTGGTGCAGATATTATTGCCGGAAGA
ACCAATTTGGAAGAATTGGAAAAATATGCTTTGGAACTTCCTCCAGTTGA
ACCGCATCCTGGTAAACAAGAGTATCTGGAAGCTGTTTTCAACAACGTAA
TGTTTACAGTTTAATAA (SEQ ID NO: 9)
MVNGLTNIPPVKFEGRDSKKALSFKYYNPDEMIQGKKMKDYLKFAMSYWH
TLCGDGTDPFGSSTIDRDYSGQTPMEKAKTKADVAFALMQILGIEYFCFH
DLDIAPTGNSLKELKNNLIEITDYIKGLMDKTGIKLLWGTANCFSHPRYM
NGAGTSPQADIFACAAAQIKNAIDATIKLGGTGYVFWGGREGYETLNTNM
EIELDNMAKLMHMAVDYARSKGFTGDFYIEPKPKEPTKHQYDFDVATVVG
FLRKYGLDKDFKMNIEANHATLAGHTFQHELNVARVNNVFGSIDANQGDL
LLGWDTDQFPTNVYDTTLCMLEVIKAGGFTNGGLNFDAKVRRASYTMEDI
ILAYISGMDTFALGLKIANKIIEDGRIDEFVSRRYASYKTGIGADIIAGR
TNLEELEKYALELPPVEPHPGKQEYLEAVFNNVMFTV (SEQ ID NO: 10)
ATGAGGGAACATTTCTTGGAAATCAATAAAATCAAATTCGAAGGTGGAGA
CTCCACAAATCCTTTGGCTTTTAAATATTATGATGCTAATAGAATAGTCG
CCGGTAAGAAAATGAAGGATCATTTGAGATTCGCTCTATCCTATTGGCAC
ACTTTGACTGGCAATGGTACTGATCCATTCGGACAACCTACTATGGAAAG
GGACTACAATAGCCTTGACGGAATTGAACTATCTAAGGCCAGAGTTGATG
CTGCTTTCGAACTGATGACTAAGCTGGGCATCGAGTTCTTCTGCTTCCAT
GATTTGGACATTGCTCCAGAAGGTAACTCCTTGCAAGAGAAATTGGATAA
TCTGGACACGATTCTTGAAAGAATCGAGGATAAGATGAAAGAAACCGGCA
TCAAGTGTTTATGGGGCACTACTAACGCCTTTAGTCACCCCAGGTTCATG
CACGGTGCAGCAACTTCTCCTAATGCCGATGTTTTCGCTTTTGCTGCTGC
GCAAGTAAAGAAAGCATTAGAAATTACCCACAGGTTGAGAGGTGAGAATT
ATGTCTTTTGGGGTGGTAGAGAAGGTTACGAGACCCTGCTGAATACTGAC
ATTGCCTTAGAAAATGACAACTTGGCTAAGTTTTTGAAGATGGCCAAAGA
TTACGCTAGGAATATTGGATTCGAAGGTCAATTCTTGATCGAGCCTAAAC
CCAAGGAACCTACTAAGCACCAGTACGACTTCGACACTATGACCGTATTA
GGTTTTTTAAGGAAGTACAATTTGATAGATGACTTCAAGTTGAACATCGA
AGCCAATCACGCCACACTAGCAGGCCACACATTCCAGCATGAGTTAGCTA
TGGCTAGGATAAACGGTGTATTGGGTTCTGTTGATGCTAACCAAGGTGAC
CTATTGTTAGGATGGGACACGGATCAATTCCCCACAAACATTTATGATGC
TACTCTTTCTATGTATGAGGTCTTGAAAAACGGTGGTATCGCACCTGGCG
GCCTGAACTTTGATGCGAAAGTTCGTAGGGGTTCATTCAAGCCTGACGAT
CTTTTCATTGCTTACATTGTTGGTATGGATACTTTCGCGAAAGGGTTACT
TGTAGCTGATAAATTGCTTACTGACGGTGTACTTGAAAATTTTGTGACTA
AAAGGTATGAATCTTACACTGCTGGTATTGGTAAAAAGATCATTGAAGAT
GCTACCTCTTTTGAAGAATTGGCAGAATATGCTTTGAAACATGATAAAAT
TGTCTTGGAATCTGGTAGACAAGAGATGCTGGAAGATATTGTCAACAGAT
ATATTTATAAATAATAA (SEQ ID NO: 11)
MREHFLEINKIKFEGGDSTNPLAFKYYDANRIVAGKKMKDHLRFALSYWH
TLTGNGTDPFGQPTMERDYNSLDGIELSKARVDAAFELMTKLGIEFFCFH
DLDIAPEGNSLQEKLDNLDTILERIEDKMKETGIKCLWGTTNAFSHPRFM
HGAATSPNADVFAFAAAQVKKALEITHRLRGENYVFWGGREGYETLLNTD
IALENDNLAKFLKMAKDYARNIGFEGQFLIEPKPKEPTKHQYDFDTMTVL
GFLRKYNLIDDFKLNIEANHATLAGHTFQHELAMARINGVLGSVDANQGD
LLLGWDTDQFPTNIYDATLSMYEVLKNGGIAPGGLNFDAKVRRGSFKPDD
LFIAYIVGMDTFAKGLLVADKLLTDGVLENFVTKRYESYTAGIGKKIIED
ATSFEELAEYALKHDKIVLESGRQEMLEDIVNRYIYK (SEQ ID NO: 12)
ATGGAATTTTTCAAAGGCATTGATAAAGTCAAATACGAAGGTGTAAAAAC
CAATAATCTTTTGGCTTTTGCACATTATAATCCTGAAGAAGTAATCTTGG
GTAAGAAAATGAAGGATCATTTGAAATTCGCTATGTCCTATTGGCACACT
TTGACTGGCGAAGGTACTGATCCATTCGGAAATGCTACTATGGACAGGGA
ATGGAATGAATATACCCCAATGGAAAAAGCTAAGGCCAGAGTTAAAGCTG
GTTTCGAATTTATGGAAAAGCTGGGCTTGGAGTACTTCTGCTTCCATGAT
AAAGACATTGCTCCAGAAGCTGAAACCTTGGAAGAGTACCATAGGAATCT
GGACGAGATTGTTGATTTGATCGAGGAGGAGATGAAAAGAACCGGCATCA
AGTTGTTATGGGGCACTTCTAACATGTTTAGTCACCCCAGGTTCATGCAC
GGTGCAGCAACTTCTTGTAATGCCGATGTTTTCGCTTATGCTGCTGCGCA
AACAAAGAAAGCATTAGAAATTACCAAAAGGTTGAACGGTACGGGTTATG
TCTTTTGGGGTGGTAGAGAAGGTTACGAGACCCTGCTGAATACTGACATT
GGCTTAGAACTGGACAACTTGGCTAGGTTTTTGCAGATGGCCGTAGATTA
CGCTAAGAAAATTGGATTCGAAGGTCAATTCTTCATCGAGCCTAAACCCA
AGGAACCTACTAAGCACCAGTACGACTTCGACACTACTACCGTATTAGAA
TTTTTAAGGAAGTACAATTTGGATAAATACTTCAAGATGAACATCGAAGC
CAATCACGCCACACTAGCAGGCCACACATTCCAGCATGAGTTATGTACGG
CTAGGATAAACGGTGTATTCGGTTCTATTGATGCTAACCAAGGTGACATG
TTGTTAGGATGGGACACGGATCAATTCCCCACAAACGTTTATGATGCTGT
TCTTGCTATGTATGAGACCTTGTTAGCCGGTGGTTTCAAAGAAGGCGGCC
TGAACTTTGATGCGAAAGTTCGTAGGGGTTCATTCGAGCCTAAAGATTTG
TTCTATGCTTACATTTCTGGTATGGATACTTTCGCGAAAGGGTTAAAAGT
AGCTGCTAAATTGATTGAAGACGGTACATTTGAAAAGATTAAGGTTGAAA
GGTATTCCTCTTACACGACCGGTATTGGTAAACAGATCGTTAACGGAGAA
GTCGGTTTTGAAGAATTGTCAAAATATGCTTTGACTAATGGTGTAAAAAA
AAATTCATCTGGTAGACAAGAGATGCTGGAAAATATTTTGAACAGATATA TTTATGAATAATAA
(SEQ ID NO: 13) MEFFKGIDKVKYEGVKTNNLLAFAHYNPEEVILGKKMKDHLKFAMSYWHT
LTGEGTDPFGNATMDREWNEYTPMEKAKARVKAGFEFMEKLGLEYFCFHD
KDIAPEAETLEEYHRNLDEIVDLIEEEMKRTGIKLLWGTSNMFSHPRFMH
GAATSCNADVFAYAAAQTKKALEITKRLNGTGYVFWGGREGYETLLNTDI
GLELDNLARFLQMAVDYAKKIGFEGQFFIEPKPKEPTKHQYDFDTTTVLE
FLRKYNLDKYFKMNIEANHATLAGHTFQHELCTARINGVFGSIDANQGDM
LLGWDTDQFPTNVYDAVLAMYETLLAGGFKEGGLNFDAKVRRGSFEPKDL
FYAYISGMDTFAKGLKVAAKLIEDGTFEKIKVERYSSYTTGIGKQIVNGE
VGFEELSKYALTNGVKKNSSGRQEMLENILNRYIYE (SEQ ID NO: 14)
ATGAGTGAAGTTTTCTCCGGCATCTCAAATATCAAATTCGAAGGTTCAGG
CTCCGATAATCCTTTGGCTTTTAAATATTATGATCCTAAAGCAGTAATCG
GCGGTAAGACTATGGAAGAGCATTTGAGATTCGCTGTAGCCTATTGGCAC
ACTTTTGCTGCCCCTGGTGCTGATATGTTCGGAGCAGGTTCTTATGTCAG
GCCCTGGAATACCATGTCCGACCCATTGGAAATAGCTAAGTACAAAGTTG
AAGCTAATTTCGAATTTATTGAAAAGCTGGGCGCCCCGTTCTTCGCCTTC
CATGATAGAGACATTGCTCCAGAAGGTGACACCTTGGCAGAGACCAATAA
GAATCTGGACACGATTGTTTCTGTCATCAAGGATAGGATGAAATCATCCC
CCGTCAAGTTGTTATGGGGCACTACTAACGCCTTTGGTAACCCCAGGTTC
ATGCACGGTGCATCAACTTCTCCTAATGCCGATATTTTCGCTTATGCTGC
TGCGCAAGTAAAGAAAGCTATGGAAATTACCAAAGAGTTGGGCGGTGAGA
ATTATGTCTTTTGGGGTGGTAGAGAAGGTTACGAGACCCTGCTGAATACT
GACATGAAATTAGAACTGGACAACTTGGCTAGGTTTTTGAAGATGGCCGT
AGATTACGCTAAGGAAATTGGATTCGACGGTCAATTCTTGATCGAGCCTA
AACCCAAGGAACCTACTAAGCACCAGTACGACTTCGACACTGCTACCGTA
ATAGGTTTTTTAAAGACGTACGGGTTGGACCCCTACTTCAAGATGAACAT
CGAAGCCAATCACGCCACACTAGCAGGCCACACATTCCAGCATGAGTTAG
CTATGTGTAGGATAAACGATATGTTGGGTTCTATTGATGCTAACCAAGGT
GACGTAATGTTAGGATGGGACACGGATCAATTCCCCACAAACCTTTATGA
TGCTACTCTTGCTATGGTGGAGGTCTTGAAAGCCGGTGGTTTGAAAAAAG
GCGGCCTGAACTTTGATTCTAAAGTTCGTAGGGGTTCATTCGAGCCTTCC
GATCTTTTCTATGGTCACATTGCTGGTATGGATACTTTCGCGAAAGGGTT
AATTATAGCTAATAAAATCGTTGAAGACGGTAAATTTGATGCTTTTGTGG
CTGACAGGTATTCCTCTTACACGAACGGTATTGGTAAAGATATTGTTGAA
GGAAAAGTCGGTTTTAAAGAATTGGAACAATATGCTTTGACTGCTAAAAT
TCAAAATAAATCTGGTAGACAAGAGATGCTGGAAGCTCTTTTGAACCAAT
ATATTTTGGAAACTAAATAATAA (SEQ ID NO: 15)
MSEVFSGISNIKFEGSGSDNPLAFKYYDPKAVIGGKTMEEHLRFAVAYWH
TFAAPGADMFGAGSYVRPWNTMSDPLEIAKYKVEANFEFIEKLGAPFFAF
HDRDIAPEGDTLAETNKNLDTIVSVIKDRMKSSPVKLLWGTTNAFGNPRF
MHGASTSPNADIFAYAAAQVKKAMEITKELGGENYVFWGGREGYETLLNT
DMKLELDNLARFLKMAVDYAKEIGFDGQFLIEPKPKEPTKHQYDFDTATV
IGFLKTYGLDPYFKMNIEANHATLAGHTFQHELAMCRINDMLGSIDANQG
DVMLGWDTDQFPTNLYDATLAMVEVLKAGGLKKGGLNFDSKVRRGSFEPS
DLFYGHIAGMDTFAKGLIIANKIVEDGKFDAFVADRYSSYTNGIGKDIVE
GKVGFKELEQYALTAKIQNKSGRQEMLEALLNQYILETK (SEQ ID NO: 16)
ATGAAGAACTATTTCCCCAACGTCCCAGAAGTCAAATACGAAGGTCCAAA
CTCCACAAATCCTTTCGCTTTTAAATATTATGATGCTAATAAAGTAGTCG
CCGGTAAGACCATGAAGGAGCATTGTAGATTCGCTCTATCCTGGTGGCAC
ACTTTGTGTGCCGGTGGTGCTGATCCATTCGGAGTAACTACTATGGACAG
GACCTACGGTAACATTACCGACCCAATGGAACTAGCTAAGGCCAAAGTTG
ATGCTGGTTTCGAACTGATGACTAAGCTGGGCATCGAGTTCTTCTGCTTC
CATGATGCCGACATTGCTCCAGAAGGTGACACCTTCGAAGAGTCCAAGAA
GAATCTGTTCGAGATTGTTGATTACATCAAGGAGAAGATGGACCAAACCG
GCATCAAGTTGTTATGGGGCACTGCTAACAACTTTAGTCACCCCAGGTTC
ATGCACGGTGCATCAACTTCTTGTAATGCCGATGTTTTCGCTTATGCTGC
TGCGAAAATAAAGAACGCTTTAGATGCGACCATCAAGTTGGGCGGTAAGG
GTTATGTCTTTTGGGGTGGTAGAGAAGGTTACGAGACCCTGCTGAATACT
GACCTGGGCTTAGAACTGGACAACATGGCTAGGCTAATGAAGATGGCCGT
AGAATACGGTAGGGCTAATGGATTCGACGGTGACTTCTACATCGAGCCTA
AACCCAAGGAACCTACTAAGCACCAGTACGACTTCGACACTGCTACCGTA
TTAGCTTTTTTAAGGAAGTACGGGTTGGAAAAAGACTTCAAGATGAACAT
CGAAGCCAATCACGCCACACTAGCAGGCCACACATTCGAGCATGAGTTAG
CTATGGCTAGGGTAAACGGTGCATTCGGTTCTGTTGATGCTAACCAAGGT
GACCCAAACTTAGGATGGGACACGGATCAATTCCCCACAGACGTTCATTC
TGCTACTCTTGCTATGCTGGAGGTCTTGAAAGCCGGTGGTTTCACAAATG
GCGGCCTGAACTTTGATGCGAAAGTTCGTAGGGGTTCATTCGAGTTTGAC
GATATTGCCTATGGTTACATTGCTGGTATGGATACTTTCGCGTTAGGGTT
AATTAAAGCTGCTGAAATCATTGATGACGGTAGAATTGCCAAGTTTGTGG
ATGACAGGTATGCCTCTTACAAGACCGGTATTGGTAAAGCGATCGTTGAC
GGAACTACCTCTTTGGAAGAATTGGAACAATACGTGTTGACTCATTCTGA
ACCTGTCATGCAATCTGGTAGACAAGAGGTTCTGGAAACTATTGTCAACA
ACATATTGTTTAGATAATAA (SEQ ID NO: 17)
MKNYFPNVPEVKYEGPNSTNPFAFKYYDANKVVAGKTMKEHCRFALSWWH
TLCAGGADPFGVTTMDRTYGNITDPMELAKAKVDAGFELMTKLGIEFFCF
HDADIAPEGDTFEESKKNLFEIVDYIKEKMDQTGIKLLWGTANNFSHPRF
MHGASTSCNADVFAYAAAKIKNALDATIKLGGKGYVFWGGREGYETLLNT
DLGLELDNMARLMKMAVEYGRANGFDGDFYIEPKPKEPTKHQYDFDTATV
LAFLRKYGLEKDFKMNIEANHATLAGHTFEHELAMARVNGAFGSVDANQG
DPNLGWDTDQFPTDVHSATLAMLEVLKAGGFTNGGLNFDAKVRRGSFEFD
DIAYGYIAGMDTFALGLIKAAEIIDDGRIAKFVDDRYASYKTGIGKAIVD
GTTSLEELEQYVLTHSEPVMQSGRQEVLETIVNNILFR
Example 2
Transformation and Growth of Transformed Strains on Xylose
Plasmids PLS4420 (polynucleotide encoding Ruminococcus flavefaciens
xylose isomerase), PLS1569 (polynucleotide encoding Clostridium
phytofermentans xylose isomerase) and PLS1567 (vector control with
no xylose isomerase polynucleotide) were used to transform S.
cerevisiae BY4741 (MATa; his3.DELTA.1 leu2.DELTA.0 met15.DELTA.0
ura3.DELTA.0). Transformants were selected on YPD plates (Difco YPD
agar containing yeast extract 10 g/L; peptone 20 g/L; dextrose 20
g/L; agar 15 g/L) supplemented with G418 antibiotic (200 ug/ml).
Positive transformants were confirmed using PCRdiagnostics.
S. cerevisiae BY4741 strain transformed with plasmids PLS4420
(containing the polynucleotide encoding Ruminococcus flavefaciens
xylose isomerase (SEQ ID NO:3)), PLS1567 (empty vector) and PLS1569
(containing the polynucleotide encoding Clostridium phytofermentans
xylose isomerase (SEQ ID NO:16)) were grown on YPD plates for 48
hrs at 30.degree. C. Single colonies were used to inoculate 400 ul
of YPD liquid culture (Difco YPD Broth containing 10 g/L yeast
extract, 20 g/L peptone and 20 g/L dextrose) supplemented with 200
ug/ml G418. Cells were grown at 30.degree. C. for 24 h at 250 rpm.
This culture was used to inoculate YP (10 g/L yeast extract, 20 g/L
peptone) media containing 0.5% glucose and 4% xylose at 12.5%
starting inoculum. Cultures were incubated at 30.degree. C. and 250
rpm. Growth was monitored by measuring the optical density at 600
nm. After 72 h, the residual xylose was measured using a
spectrophotometric assay (Megazyme xylose assay; Cat no. K-XYLOSE)
performed according to the manufacture's protocol. The results are
shown in Table 2-1.
TABLE-US-00008 TABLE 2-1 Growth and Xylose Consumption for
Ruminococcus flavefaciens Xylose Isomerase Xylose remaining Strain
OD 600 nm (OD 340 nm) BY4741-PLS1567 7.93 .+-. 0.14 0.46 .+-. 0.01
Vector Control BY4741-PLS1569 11.31 .+-. 0.49 0.39 .+-. 0.01
Transformed with the polynucleotide encoding the Clostridium
phytofermentans xylose isomerase (SEQ ID NO: 17) BY4741-PLS4420
8.99 .+-. 0.84 0.42 .+-. 0.02 Transformed with the polynucleotide
encoding the Ruminococcus flavefaciens xylose isomerase (SEQ ID NO:
3)
The results indicate that strains transformed with PLS4420
(polynucleotide encoding Ruminococcus flavefaciens xylose
isomerase) and PLS1569 (polynucleotide encoding Clostridium
phytofermentans xylose isomerase) consumed greater amounts of
xylose and grew to higher ODs than the control strain transformed
with PLS1567 (vector control) due to the activity of the xylose
isomerases present.
Example 3
Activity of Xylose Isomerase in Additional Yeast Strains
PLS4420 (polynucleotide encoding Ruminococcus flavefaciens xylose
isomerase), PLS1569 (polynucleotide encoding Clostridium
phytofermentans xylose isomerase) and PLS1567 (vector control) were
used to transform S. cerevisiae NRRL YB-1952 (ARS culture
collection) and S. cerevisiae Superstart (LYCC6469; Lallemand).
Single colonies of transformed strains were used to inoculate 400
ul of YPD medium containing 200 ug/ml G418. The cultures were grown
at 30.degree. C. for 24 hrs at 250 rpm. These cultures were used to
inoculate 400 ul of YP containing 4% xylose supplemented with 200
ug/ml of G418. Cultures were grown in a Dasgip BioLector
Microreactor system at 30.degree. C., 95% relative humidity, and
800 rpm.
Strains transformed with PLS4420 (polynucleotide encoding
Ruminococcus flavefaciens xylose isomerase) and PLS1569
(polynucleotide encoding Clostridium phytofermentans xylose
isomerase) grew to significantly higher ODs than the control strain
with PLS1567 (vector control) in the xylose-containing medium due
to the activity of the xylose isomerases present. This data are
provided in FIG. 7, which shows a plot of Absorbance Units as a
function of time, where absorbance correlates to cell growth.
Example 4
Activity of Homologous Xylose Isomerases
Plasmids PLS1567 (vector control), PLS1569 (polynucleotide encoding
Clostridium phytofermentans xylose isomerase, SEQ ID NO:16),
PLS4420 (polynucleotide encoding Ruminococcus flavefaciens xylose
isomerase, SEQ ID NO: 3), PLS4416 (polynucleotide encoding
Alkaliphilus metalliredigens QYMF xylose isomerase, SEQ ID NO:10),
PLS4417 (polynucleotide encoding Fusobacterium mortiferum xylose
isomerase ATCC 9817 xylose isomerase, SEQ ID NO:12), PLS4418
(polynucleotide encoding Epulopiscium sp. 'N.t. morphotype B xylose
isomerase, SEQ ID NO:8) and PLS4419 (polynucleotide encoding
Clostridium cellulolyticum H10 xylose isomerase, SEQ ID NO:14) were
transformed into strain S. cerevisiae Superstart LYCC6469
(Lallemand) and the cultures were evaluated for growth on
xylose-based media as described in Example 2. As described in
Example 1, all polynucleotides encoding the xylose isomerases were
codon-optimized for expression in yeast. Growth was measured by
monitoring OD at 600 nm at 96 hrs. Residual xylose was measured
using a spectrophotometric assay (Megazyme xylose assay; Cat no.
K-XYLOSE) performed according to the manufacturer's protocol. The
results are shown in Table 4-1.
TABLE-US-00009 TABLE 4-1 Xylose Utilized by S. cerevisiae
Transformed with Polynucleotides Encoding Homologues of
Ruminococcus flavefaciens Xylose Isomerase Sequence Identity to SEQ
ID NO: 2 SEQ ID NOS: Source of Xylose (Ruminococcus Xylose
Remaining (polynucleotide)/ Isomerase Gene in flavefaciens Xylose
(Absorbance at (polypeptide) S. cerevisiae Isomerase) 340 nm) SEQ
ID NOS: 10 & 11 Plasmid PLS4416 - 59.8% 0.41 .+-. 0.002
Alkaliphilus metalliredigens SEQ ID NOS: 12 & 13 Plasmid
PLS4417 - 62.4% 0.41 .+-. 0.003 Fusobaceterium moriferem - SEQ ID
NOS: 8 & 9 Plasmid PLS4418 - 65.7% 0.41 .+-. 0.004 Epulopiscium
sp. `N.t. morphotype B` SEQ ID NOS: 14 & 15 Plasmid PLS4419 -
60.0% 0.41 .+-. 0.004 Clostridium cellulolyticum H10 SEQ ID NOS: 16
& 17 PLS1569 - Clostridium 64.6% 0.36 .+-. 0.006
phytofermentans PLS1567 - Vector 0 0.41 .+-. 0.002 control SEQ ID
NOS: 3 & 4 PLS4420 - 100% 0.39 .+-. 0.001 Ruminococcus
flavefaciens
Strains transformed with PLS4420 (polynucleotide encoding
Ruminococcus flavefaciens xylose isomerase) and PLS1569
(polynucleotide encoding Clostridium phytofermentans xylose
isomerase) showed significantly greater xylose utilization compared
to the control strain with plasmid PLS1567 whereas all other
strains had growth and xylose utilization similar to the control
strain.
Table 4-2 provides the sequence identity of the Ruminococcus
flavefaciens xylose isomerase to various xylose isomerases.
Expression of these xylose isomerases was evaluated in
Saccharomyces cerevisiae.
TABLE-US-00010 TABLE 4-2 Sequence Identities of Various Xylose
Isomerases (Expressed From S. cerevisiae in the literature)
Relative to the Ruminococcus flavefaciens Xylose Isomerase Sequence
Identity Compared to Accession No. Source SEQ ID NO: 2 Reference
ABX41597.1 Clostridium phytofermentans ISDg 64.6% Brat et al. 2009
Q9FKK7.2 Arabidopsis thaliana 52.4% Brat et al. 2009 AAU42385.1
Bacillus licheniformis ATCC 14580 18.8% Brat et al. 2009 ABE33371.1
Burkholderia xenovorans LB400 49.7% Brat et al. 2009 AAA25258.1
Lactobacillus pentosus 51.6% Brat et al. 2009 AAT88354.1 Leifsonia
xyli subsp. xyli str. CTCB07 24.0% Brat et al. 2009 AAZ36203.1
Pseudomonas syringae pv. Phaseolicola 48.9% Brat et al. 2009 1448A
P27157.1 Staphylococcus xylosus 54.6% Brat et al. 2009 1QT1
Streptomyces Diastaticus 28.5% Brat et al. 2009 AAM43321.1
Xanthomonas campestris pv. Campestris 53.1% Brat et al. 2009 str.
ATCC 33913 ACY90798.1 Salmonella enteric subsp. enteric 50.1% Brat
et al. 2009 serovar typhimurium str. 14028S AAK88959.2
Agrobacterium tumefaciens str. C58 48.0% Brat et al. 2009
YP_527976.1 Saccharophagus degradans 2-40 50.7% Brat et al. 2009
YP_003193917.1 Robiginitalea biformata HTCC2501 54.5% Brat et al.
2009 CAB76571.1 Piromyces sp. E2 48.9% Kuyper et al. 1993 1BXG
Thermus thermophilus 21.4% Walfridsson et al. 1996 A8A623.1
Escherichia coli HS 50.6% Chan et al. 1989
Example 5
Fermentation Activity of Xylose Isomerase
Plasmids PLS1567 (vector control), PLS1569 (polynucleotide encoding
Clostridium phytofermentans xylose isomerase, SEQ ID NO:16) and
PLS4420 (polynucleotide encoding Ruminococcus flavefaciens xylose
isomerase, SEQ ID NO: 3) were transformed into strain S. cerevisiae
BY4741 and S. cerevisiae Superstart LYCC6469 (Lallemand). Single
colonies of transformed strains were used to inoculate 400 ul of
YPD medium containing 1 mM MgSO.sub.4 and 200 ug/ml G418. The
cultures were grown at 30.degree. C. for 72 hrs at 250 rpm. Then,
40 .mu.l of saturated cultures were used to inoculate 400 ul of YPD
containing 2% xylose supplemented with 1 mM MgSO.sub.4 and 200
ug/ml of G418. The cultures were grown at 30.degree. C. for 48 hrs
with 250 rpm shaking. At 48 hrs, the cells were spun down at
22.degree. C. for 10 mins.
To assay for fermentation, cells were re-suspended in 400 ul of YPD
containing 4% xylose supplemented with 1 mM MgSO.sub.4 and 200
ug/ml of G418. The plates were sealed with mats and incubated at
30.degree. C. with 160 rpm shaking. At 120 hrs, cells were
harvested. The residual sugars and ethanol in the supernatant were
measured using HPLC, as known in the art (See e.g., DuPont et al.,
Carbohydr. Polym., 68:1-16 [2007], which is incorporated herein by
reference). In some experiments, the residual xylose in the
supernatant was measured using a spectrophotometric assay (e.g.,
Megazyme xylose assay; Cat no. K-XYLOSE) performed according to the
manufacture's protocol. As indicated in FIG. 8, strains transformed
with PLS1569 and PLS4420 consumed higher amounts of xylose compared
to the vector control in both strains.
Example 6
Xylose Utilization by Xylose Isomerase Variants
The Ruminococcus flavefaciens xylose isomerase was subjected to
directed evolution to improve xylose utilization activity.
Libraries of Ruminococcus flavefaciens xylose isomerase variants
were screened as described in Example 2 for xylose-based growth.
Libraries for the first of two iterative rounds of directed
evolution were constructed by random mutagenesis of the degenerate
third position of DNA codons (See e.g., Le Calvez et al., Gene
170:51-55 [1996], which is incorporated herein by reference), and
saturation mutagenesis of specific amino acids performed by
automated parallel synthesis of polynucleotide variants as
described in WO 2010/144103. Libraries for the second round of
evolution were constructed by semi-synthetic DNA shuffling as known
in the art (See e.g., Stutzman-Engwall et al, Metab. Eng., 7:27-37
[2005]) to recombine beneficial mutations observed in round 1 of
directed evolution and test additional mutations. The complete list
of improved variants and corresponding sequences is provided in
Tables 6-1 and 6-2. All mutations are with reference to the
starting Ruminococcus flavefaciens xylose isomerase gene construct
and activity fold improvements over positive control (FIOPC) are
with reference to the backbone for each round of evolution: the
starting Ruminococcus flavefaciens xylose isomerase gene construct
for round 1 and variant 2 for round 2. In these Tables, the results
are provided as values .gtoreq.1 (i.e., 1-1.4), .gtoreq.1.5 (i.e.,
1.5-1.9), .gtoreq.2 (i.e., 2-2.4), .gtoreq.2.5 (i.e., 2.5-2.9),
.gtoreq.3 (i.e., 3-3.4), .gtoreq.3.5 ((i.e., 3.5-3.9), or
.gtoreq.4.
TABLE-US-00011 TABLE 6-1 R. flavefaciens Xylose Isomerase Rd1
Improved Variants FIOPC VARIANT Xylose NO: Active Mutations Silent
Mutations Used 1 1.0 2 E372G
c138a/t150a/g783a/t1143g/c1146t/c1155a/t1263a/a1269g .gtoreq.3 3
N433R .gtoreq.2 4 F250C .gtoreq.2 5 G62F .gtoreq.2 6 t1263a/t1266g
.gtoreq.1.5 7 Q424H .gtoreq.1.5 8 M199V .gtoreq.1.5 9 E414A
.gtoreq.1.5 10 G390M .gtoreq.1.5 11 V431E .gtoreq.1.5 12 A400G
.gtoreq.1.5 13 Q116C .gtoreq.1.5 14
t261a/t309g/t312g/t429c/c432t/c435t/a903g/a906g .gtoreq.1.5 15
V247A t120c/t360a/c993a/c996g/g999a .gtoreq.1.5 16
a180t/c291t/c294t/a693g/c696t/a813g/a816t/c822t/a825g .gtoreq.1.5
17 I391L .gtoreq.1.5 18
g123a/a333g/t403c/c423t/t426c/t429c/c435a/c549g/ .gtoreq.1.5
t552c/t981g/c984t/a987g/t990c/a1221g 19 Q70E .gtoreq.1.5 20 H274R
c213t/a219g/c339a/a888g/t891g/c894t/a897g/g1011t/ .gtoreq.1.5
t1017a 21 t66a/c138g/t150g/a258g/t261c/t267c/t543g/t546c/c549t
.gtoreq.1.5 22 g351t/c354t/t360g/c600g; t834c/a840g .gtoreq.1.5 23
T18C .gtoreq.1.5 24 c51a/a54g/g1011a .gtoreq.1.5 25
a318g/t558a/t561a/a567g/t570g/t735g/c798g/t801c/ .gtoreq.1
c807g/a810g 26 T64Q .gtoreq.1 27 F328H .gtoreq.1 28
c213g/a219g/a225g/c411g/t414c/t417g/g528a/g531a/ .gtoreq.1
c534g/a819g/a825g 29 F360M .gtoreq.1 30 T236A .gtoreq.1 31
g123a/a126g/c129t/t132a/a135c/t1164c/c1167t/t1170g .gtoreq.1 32
a1125g .gtoreq.1 33 t66c/c138g/g582a/a987g .gtoreq.1 34 M206T
a1086g/a1095g .gtoreq.1 Q277R 35 g654a/t657g .gtoreq.1 36 M199A
.gtoreq.1 37 t9c/c12t/c15t/g123a/t132g/a135g/t492a/a606g/c612t
.gtoreq.1 38 g222t/a225g/a453t/t462g/t465g/g528a/g531a/c534g/
.gtoreq.1 t537g/c579g/a693g/c696t/a774g/c780t/g1134a/g1140a 39
E2S/Q13K .gtoreq.1 40 N6G .gtoreq.1 41 c108g .gtoreq.1 42 N330G
.gtoreq.1 43 E29N .gtoreq.1 44 I371Q .gtoreq.1 45
a93t/c96t/t102c/a180g/g768a/t1008c/g1011t/a1014g/ .gtoreq.1 t1017g
46 N433A .gtoreq.1 47 S386K .gtoreq.1 48 t168a/c177t/a420g
.gtoreq.1 49 Y182C/ a126g/t132c/a135c/g438a/c441t/c447t/t450c
.gtoreq.1 G356A 50
a54g/t60a/t168c/t171c/c177t/a180t/c213a/c216t/a219c/ .gtoreq.1
g222a/a225c/t891a/c894t/a897c 51 K127I .gtoreq.1 52 D382G/
.gtoreq.1 N433H 53 t168g/a819g/c822t/a825g .gtoreq.1 54 I371L
.gtoreq.1 55 a516g/t558g/a564g/c798g/c804t/a810c/a1209t/a1212c
.gtoreq.1 56 S109D .gtoreq.1 57 t1065c .gtoreq.1 58 L248S
t414g/t417g/a420g/a453c/t459a/t462c/c822t/a825t/ .gtoreq.1
t1008c/t1017g/t1020g 59
c138a/c147t/t186c/g192t/c858t/t861g/a864g/a987t .gtoreq.1 60 T18L
.gtoreq.1 61 N330W .gtoreq.1 62 R281L .gtoreq.1 63 V434S .gtoreq.1
64 N330Y .gtoreq.1 65 S71L .gtoreq.1 66 G398E .gtoreq.1 67 G342P
.gtoreq.1 68 T18K/ .gtoreq.1 N330L 69 T67S .gtoreq.1 70 V380W
.gtoreq.1 71 R423G a54g/g438a/c447t/t450g/c798t/t801c/c804t/c807a
.gtoreq.1 72 I371R .gtoreq.1 73 K233C .gtoreq.1 74 R375V .gtoreq.1
75 a48g/c108a/t882c .gtoreq.1 76 I371T .gtoreq.1 77 t1137c
.gtoreq.1 78 a816t/a819g/c822t/g1011t/a1014g .gtoreq.1 79 S404Y
.gtoreq.1 80 g528a/t537a/c573t/c579g/g585c/c696a/t705g .gtoreq.1 81
c15g/t132a/t249a/t252g/c927g/a930g/t1290c .gtoreq.1 82
t546c/c549t/c858t/t861g/a864c/t870a .gtoreq.1 83
c288t/c291t/c294t/t300c/a405g/t651c .gtoreq.1 84 T236L .gtoreq.1 85
I391A .gtoreq.1 86 K407L .gtoreq.1 87 N6H .gtoreq.1 88 c307t
.gtoreq.1 89 F250V .gtoreq.1 90 K111L .gtoreq.1 91 G342V .gtoreq.1
92 K78R a93t/c96t/t102g/a180t/a813g/a819g/a825t .gtoreq.1 93 V434Q
.gtoreq.1 94 T329S .gtoreq.1 95 E372T/ .gtoreq.1 K399V 96 K378A
.gtoreq.1 97 a48g/c51t/a54g/t57c/t60g/a1209g .gtoreq.1 98 T389H
.gtoreq.1 99 M426R .gtoreq.1 100 D382N/
t168c/a180g/a813g/a816c/a819g/c822t/a825g/g1011a/ .gtoreq.1 A393T
a1014g/t1017a/t1020c 101 T18M .gtoreq.1 102 T244S t591g/c600g/a840g
.gtoreq.1 103 L435S
t102c/c213a/c216t/a219g/g222t/a225c/a813g/a819g/ .gtoreq.1
c822t/a825g 104 T18L .gtoreq.1 105 t156c/t165c .gtoreq.1 106
c411t/t414g/t417g/a420g/t429c .gtoreq.1 107
c276t/t279c/c285t/a606g/c828t/a840g/t873a/t882g/c885t .gtoreq.1 108
c480t/c522g/t708g/c720t/c762tt960c/t1228c .gtoreq.1 109 R375Q
.gtoreq.1 110 D373G c129t/a135g/c441t .gtoreq.1 111
t771c/a774g/c894t/a897g/t1128a/c1131t/t1185c .gtoreq.1 112 A339R
t211a .gtoreq.1 113 t150g/c1146t/t1152c/c1155g .gtoreq.1 114 A74G
.gtoreq.1 115 c108t/c396t/t402c .gtoreq.1 116 R375T .gtoreq.1 117
V81I .gtoreq.1 118
t261a/t543g/t552c/a741c/t870g/t960c/t1026a/a1029t/ .gtoreq.1
c1032t/g1035c 119 c441t/c447t/a810c/a1095g .gtoreq.1 120 a228g
.gtoreq.1 121 F436G .gtoreq.1 122 N433R .gtoreq.1 123 A397L
.gtoreq.1 124 Q128A .gtoreq.1 125 S156T .gtoreq.1
TABLE-US-00012 TABLE 6-2 R. flavefaciens Xylose Isomerase Rd2
Improved Variants FIOPC VARIANT Xylose NO: Active Mutations Silent
Mutations Used 2 E372G c138a/t150a/g783a/t1143g/c1146t/c1155a/
t1263a/a1269g 126 Q70E/E372G/G398E/
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.4.5 V434S
t1263a/a1269g 127 Q70E/S109D/T236A/
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.4 E372G/V434S
t1263a/a1269g 128 Q70E/T236A/E372G/
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.4 G398E
t1263a/a1269g 129 T67S/Q70E/S109D/
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.4 T236A/E372G/S386K
t1263a/a1269g 130 T67S/Q70E/S109D
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.4 T236A/E372G/S386K
t1263a/a1269g 131 Q70E/T236L/E372G/
c138a/t150a/c307t/g783a/t1143g/c1146t/ .gtoreq.3.5 G398E
c1155a/t1263a/a1269g 132 T67S/Q70E/T236A/
c138a/t150a/c307t/g783a/t1143g/c1146t/ .gtoreq.3.5 E372G
c1155a/t1263a/a1269g 133 Q70E/S109D/E372G
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.3.5 t1263a/a1269g
134 T67S/Q70E/S109D c138a/t150a/c307t/g783a/t1143g/c1146t/
.gtoreq.3.5 E372G/V434S c1155a/t1263a/a1269g 135 Q70E/E372G/V434S
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.3.5 t1263a/a1269g
136 Q70E/T236A/R281L/ c138a/t150a/g783a/t1143g/c1146t/c1155a/
.gtoreq.3.5 A325S/E372G t1263a/a1269g 137 T67S/Q70E/T236L/
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.3.5 E372G/V431E
t1263a/a1269g 138 T67S/Q70E/T236L/
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.3 E372G/S386K
t1263a/a1269g 140 T67S/Q70E/S109D/
c138a/t150a/c307t/g783a/t1143g/c1146t/ .gtoreq.3 R281L/E372G/S404Y
c1155a/t1263a/a1269g 141 T67S/Q70E/
c138a/t150a/c307t/g783a/t1143g/c1146t/ .gtoreq.2.5
S109D/E372G/S386K c1155a/t1263a/a1269g 142 Q70E/S109D/E372G/
c138a/t150a/c307t/g783a/t1143g/c1146t/ .gtoreq.2.5 V431E
c1155a/t1263a/a1269g 143 T67S/Q70E/R281L/
c138a/t150a/c307t/g783a/t1143g/c1146t/ .gtoreq.2.5 E372G/S404Y
c1155a/t1263a/a1269g 144 T67S/Q70E/S109D/
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.2.5 E372G/V431E
t1263a/a1269g 145 T67S/Q70E/E372G/
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.2.5 G390M
t1263a/a1269g 146 T67S/Q70E/A325S/
c138a/t150a/c307t/g783a/t1143g/c1146t/ .gtoreq.2.5 E372G
c1155a/t1263a/a1269g 147 T67S/Q70E/S109D/
c138a/t150a/c307t/g783a/t1143g/c1146t/ .gtoreq.2.5
E372G/I391L/S404Y c1155a/t1263a/a1269g 148 T67S/Q70E/S109D/
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.2.5
T236L/E372G/I391L/ t1263a/a1269g G398E/V434S 149 Q70E/S109D/T236A/
c138a/t150a/c307t/g783a/t1143g/c1146t/ .gtoreq.2 E372G/I391L
c1155a/t1263a/a1269g 150 Q70E/S109D/T236L/
c138a/t150a/c307t/g783a/t1143g/c1146t/ .gtoreq.2 E372G/S386K/S404Y
c1155a/t1263a/a1269g 151 T67S/Q70E/
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.2 S109D/R281L/E372G
t1263a/a1269g 152 E372G c138a/t150a/g783a/t1143g/c1146t/c1155a/
.gtoreq.2 t1263a/a1269g 153 R281L/A325S/E372G/
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.2 A397S
t1263a/a1269g 154 T67S/Q70E/A75T/
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.2 E372G
t1263a/a1269g 155 T67S/S109D/R281L/
c138a/t150a/c307t/g783a/t1143g/c1146t/ .gtoreq.2 E372G
c1155a/t1263a/a1269g 156 Q70E/S109D/E372G/
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.2 G398E
t1263a/a1269g 157 S109D/R281L/E372G
c138a/t150a/c307t/g783a/t1143g/c1146t/ .gtoreq.1.5
c1155a/t1263a/a1269g 158 S109D/T236A/R281L/
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1.5- E372G
t1263a/a1269g 159 Q70E/T236A/E372G
c138a/t150a/c307t/g783a/t1143g/c1146t/ .gtoreq.1.5
c1155a/t1263a/a1269g 160 K78R/D373G
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1.5 t1263a/a1269g
161 T67S/S109D/E372G/ c138a/t150a/c307t/g783a/t1143g/c1146t/
.gtoreq.1.5 G398E/V434S c1155a/t1263a/a1269g 162 K16E/K111A/E372G
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1.5 t1263a/a1269g
163 T67S/Q70E/R281L/ c138a/t150a/g783a/t1143g/c1146t/c1155a/
.gtoreq.1.5 E372G t1263a/a1269g 164 T67S/Q70E/E372G/
c138a/t150a/c307t/g783a/t1143g/c1146t/ .gtoreq.1.5 S386K
c1155a/t1263a/a1269g 165 S109D/T236L/R281L/
c138a/t150a/c307t/g783a/t1143g/c1146t/ .gtoreq.1.5 A325R/E372G
c1155a/t1263a/a1269g 166 T64Q/M199A/K233C/
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1.5 E372G
t1263a/a1269g 167 E372G/K399T
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1.5 t1263a/a1269g
168 T64Q/S71L/M199A/ c138a/t150a/g783a/t1143g/c1146t/c1155a/
.gtoreq.1.5 K233C/E372G/I391L t1263a/a1269g 169 T67S/R281L/A325R/
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1.5 E372G
t1263a/a1269g 170 T64Q/L91M/A139G/
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1.5
A164V/K233C/E372G t1263a/a1269g 171 R284H/E372G
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1.5 t1263a/a1269g
172 K127R/G356A/E372G c138a/t150a/g783a/t1143g/c1146t/c1155a/
.gtoreq.1.5 t1263a/a1269g 173 E372G/I391L/S404Y/
c138a/t150a/c307t/g783a/t1143g/c1146t/ .gtoreq.1.5 V434S
c1155a/t1263a/a1269g 174 V247A/L248S/G356A/
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1.5- E372G
t1263a/a1269g 175 E372G c138a/t150a/c625t/g783a/t1143g/c1146t/
.gtoreq.1.5 c1155a/t1263a/a1269g 176 L201H/E372G
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1.5 t1263a/a1269g
177 K223T/K237A/E372G/ c138a/t150a/g783a/t1143g/c1146t/c1155a/
.gtoreq.1.5- K399T/K407E t1263a/a1269g 178 T64Q/S71L/K233C/
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1.5 F360M/E372G
t1263a/a1269g 179 Q116C/M199A/K233C/
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1.5- E372G/K407L
t1263a/a1269g 180 N6G/E372G/F436G
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1 t1263a/a1269g 181
K78R/V247A/E372G c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1
t1263a/a1269g 182 T329S/N330H/E372G/
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1 R375V
t1263a/a1269g 183 T64Q/F360M/E372G
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1 t1263a/a1269g 184
K16E/K111A/E372G/ c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1
K399T t1263a/a1269g 185 V247A
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1 t1263a/a1269g 186
K233C/E372G/K407L c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1
t1263a/a1269g 187 E372G/L435S
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1 t1263a/a1269g 188
T64Q/S71L/Q116C/ c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1
M199A/F360M/E372G/ t1263a/a1269g K407R 189 K78R/E372G/K399E/
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1 R423G t1263a 190
S71L/M199A/K233C/ c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1
E372G/K407L t1263a/a1269g 191 M206T/L248S/H274R/
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1 K399E
t1263a/a1269g 192 K127R/E372G/D373G/
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1 t1263a/a1269g 193
E29N/E372G c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1
t1263a/a1269g 194 K233C/E372G/V380W
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1 t1263a/a1269g 195
E372G/K378D c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1
t1263a/a1269g 196 Y182C/V247A/G356A
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1 t1263a/a1269g 197
S71L/E372G c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1
t1263a/a1269g 198 K78R/V247A/L248S/
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1 G356A/E372G
t1263a/a1269g 199 G356A/E372G/K399E/
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1 R423G t1263a 200
K211H/E372G/K407E c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1
t1263a/a1269g 201 T64Q/K233C/F360M/
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1 E372G/K407L/Q424H
t1263a/a1269g 202 G356A/D373G
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1 t1263a/a1269g 203
E372G/K407E c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1
t1263a/a1269g 204 Q116C/M199A/F360M/
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1 E372G
t1263a/a1269g 205 M206T/L248S/E372G
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1 t1263a/a1269g 206
N330Y/E372G/F436G c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1
t1263a/a1269g 207 K233C/F360M/E372G/
c138a/t150a/c177t/g783a/t1143g/c1146t/ .gtoreq.1 V380W/Q424H
c1155a/t1263a/a1269g 208 I371G/E372G/N433A
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1 t1263a/a1269g 209
K78R/G356A/E372G c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1
t1263a/a1269g 210 G356A/E372G
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1 t1263a/a1269g 211
E372G/K378D/K399T/ c138a/t150a/g783a/t1143g/c1146t/c1155a/
.gtoreq.1 K407E t1263a/a1269g; 212 T64Q/M199A/K233C/
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1 E372G/K407L/Q424H
t1263a/a 1269g 213 T64Q/K233C/F250C/
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1 E372G
t1263a/a1269g 214 K78R/Y182C/G356A/
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1 E372G
t1263a/a1269g 215 E372G/K399T/K407E
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1 t1263a/a1269g 216
F360M/E372G/Q424H c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1
t1263a/a1269g 217 E372G/K407R
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1 t1263a/a1269g 218
T64Q/Q116C/M199A/ c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1
F360M/E372G/K407L t1263a/a1269g 219 T64Q/Q116C/K233C/
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1 E372G
t1263a/a1269g 220 T64Q/S71L/Q116C/
c138a/t150a/g783a/t1143g/c1146t/c1155a/ .gtoreq.1
K233C/F360M/E372G/ t1263a/a1269g K407L/Q424H
While particular embodiments of the present invention have been
illustrated and described, it will be apparent to those skilled in
the art that various other changes and modifications can be made
without departing from the spirit and scope of the present
invention. Therefore, it is intended that the present invention
encompass all such changes and modifications with the scope of the
present invention.
The present invention has been described broadly and generically
herein. Each of the narrower species and subgeneric groupings
falling within the generic disclosure also form part(s) of the
invention. The invention described herein suitably may be practiced
in the absence of any element or elements, limitation or
limitations which is/are not specifically disclosed herein. The
terms and expressions which have been employed are used as terms of
description and not of limitation. There is no intention that in
the use of such terms and expressions, of excluding any equivalents
of the features described and/or shown or portions thereof, but it
is recognized that various modifications are possible within the
scope of the claimed invention. Thus, it should be understood that
although the present invention has been specifically disclosed by
some preferred embodiments and optional features, modification and
variation of the concepts herein disclosed may be utilized by those
skilled in the art, and that such modifications and variations are
considered to be within the scope of this invention.
SEQUENCE LISTINGS
1
SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 19 <210>
SEQ ID NO 1 <211> LENGTH: 1317 <212> TYPE: DNA
<213> ORGANISM: Ruminococcus flavefaciens <400>
SEQUENCE: 1 atggaatttt tcagcaatat cggtaaaatt cagtatcagg gaccaaaaag
tactgatcct 60 ctctcattta agtactataa ccctgaagaa gtcatcaacg
gaaagacaat gcgcgagcat 120 ctgaagttcg ctctttcatg gtggcacaca
atgggcggcg acggaacaga tatgttcggc 180 tgcggcacaa cagacaagac
ctggggacag tccgatcccg ctgcaagagc aaaggctaag 240 gttgacgcag
cattcgagat catggataag ctctccattg actactattg tttccacgat 300
cgcgatcttt ctcccgagta tggcagcctc aaggctacca acgatcagct tgacatagtt
360 acagactata tcaaggagaa gcagggcgac aagttcaagt gcctctgggg
tacagcaaag 420 tgcttcgatc atccaagatt catgcacggt gcaggtacat
ctccttctgc tgatgtattc 480 gctttctcag ctgctcagat caagaaggct
ctcgagtcaa cagtaaagct cggcggtaac 540 ggttacgttt tctggggcgg
acgtgaaggc tatgagacac ttcttaatac aaatatggga 600 ctcgaactcg
acaatatggc tcgtcttatg aagatggctg ttgagtatgg acgttcgatc 660
ggcttcaagg gcgacttcta tatcgagccc aagcccaagg agcccacaaa gcatcagtac
720 gatttcgata cagctactgt tctgggattc ctcagaaagt acggtctcga
taaggatttc 780 aagatgaata tcgaagctaa ccacgctaca cttgctcagc
atacattcca gcatgagctc 840 cgtgttgcaa gagacaatgg tgtgttcggt
tctatcgacg caaaccaggg cgacgttctt 900 cttggatggg atacagacca
gttccccaca aatatctacg atacaacaat gtgtatgtat 960 gaagttatca
aggcaggcgg cttcacaaac ggcggtctca acttcgacgc taaggcacgc 1020
agagggagct tcactcccga ggatatcttc tacagctata tcgcaggtat ggatgcattt
1080 gctctgggct tcagagctgc tctcaagctt atcgaagacg gacgtatcga
caagttcgtt 1140 gctgacagat acgcttcatg gaataccggt atcggtgcag
acataatcgc aggtaaggca 1200 gatttcgcat ctcttgaaaa gtatgctctt
gaaaagggcg aggttacagc ttcactctca 1260 agcggcagac aggaaatgct
ggagtctatc gtaaataacg ttcttttcag tctgtaa 1317 <210> SEQ ID NO
2 <211> LENGTH: 438 <212> TYPE: PRT <213>
ORGANISM: Ruminococcus flavefaciens <400> SEQUENCE: 2 Met Glu
Phe Phe Ser Asn Ile Gly Lys Ile Gln Tyr Gln Gly Pro Lys 1 5 10 15
Ser Thr Asp Pro Leu Ser Phe Lys Tyr Tyr Asn Pro Glu Glu Val Ile 20
25 30 Asn Gly Lys Thr Met Arg Glu His Leu Lys Phe Ala Leu Ser Trp
Trp 35 40 45 His Thr Met Gly Gly Asp Gly Thr Asp Met Phe Gly Cys
Gly Thr Thr 50 55 60 Asp Lys Thr Trp Gly Gln Ser Asp Pro Ala Ala
Arg Ala Lys Ala Lys 65 70 75 80 Val Asp Ala Ala Phe Glu Ile Met Asp
Lys Leu Ser Ile Asp Tyr Tyr 85 90 95 Cys Phe His Asp Arg Asp Leu
Ser Pro Glu Tyr Gly Ser Leu Lys Ala 100 105 110 Thr Asn Asp Gln Leu
Asp Ile Val Thr Asp Tyr Ile Lys Glu Lys Gln 115 120 125 Gly Asp Lys
Phe Lys Cys Leu Trp Gly Thr Ala Lys Cys Phe Asp His 130 135 140 Pro
Arg Phe Met His Gly Ala Gly Thr Ser Pro Ser Ala Asp Val Phe 145 150
155 160 Ala Phe Ser Ala Ala Gln Ile Lys Lys Ala Leu Glu Ser Thr Val
Lys 165 170 175 Leu Gly Gly Asn Gly Tyr Val Phe Trp Gly Gly Arg Glu
Gly Tyr Glu 180 185 190 Thr Leu Leu Asn Thr Asn Met Gly Leu Glu Leu
Asp Asn Met Ala Arg 195 200 205 Leu Met Lys Met Ala Val Glu Tyr Gly
Arg Ser Ile Gly Phe Lys Gly 210 215 220 Asp Phe Tyr Ile Glu Pro Lys
Pro Lys Glu Pro Thr Lys His Gln Tyr 225 230 235 240 Asp Phe Asp Thr
Ala Thr Val Leu Gly Phe Leu Arg Lys Tyr Gly Leu 245 250 255 Asp Lys
Asp Phe Lys Met Asn Ile Glu Ala Asn His Ala Thr Leu Ala 260 265 270
Gln His Thr Phe Gln His Glu Leu Arg Val Ala Arg Asp Asn Gly Val 275
280 285 Phe Gly Ser Ile Asp Ala Asn Gln Gly Asp Val Leu Leu Gly Trp
Asp 290 295 300 Thr Asp Gln Phe Pro Thr Asn Ile Tyr Asp Thr Thr Met
Cys Met Tyr 305 310 315 320 Glu Val Ile Lys Ala Gly Gly Phe Thr Asn
Gly Gly Leu Asn Phe Asp 325 330 335 Ala Lys Ala Arg Arg Gly Ser Phe
Thr Pro Glu Asp Ile Phe Tyr Ser 340 345 350 Tyr Ile Ala Gly Met Asp
Ala Phe Ala Leu Gly Phe Arg Ala Ala Leu 355 360 365 Lys Leu Ile Glu
Asp Gly Arg Ile Asp Lys Phe Val Ala Asp Arg Tyr 370 375 380 Ala Ser
Trp Asn Thr Gly Ile Gly Ala Asp Ile Ile Ala Gly Lys Ala 385 390 395
400 Asp Phe Ala Ser Leu Glu Lys Tyr Ala Leu Glu Lys Gly Glu Val Thr
405 410 415 Ala Ser Leu Ser Ser Gly Arg Gln Glu Met Leu Glu Ser Ile
Val Asn 420 425 430 Asn Val Leu Phe Ser Leu 435 <210> SEQ ID
NO 3 <211> LENGTH: 1320 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic DNA polynucleotide codon optimized for
expression of xylose isomerase in Saccharomyces cerevisiae
<400> SEQUENCE: 3 atggaatttt tctccaacat cggaaaaatc caataccaag
gtccaaaatc cacagatcct 60 ttgtctttta aatattataa tcctgaagaa
gtaatcaacg gtaagaccat gagggagcat 120 ttgaaattcg ctctatcctg
gtggcacact atgggtggcg atggtactga tatgttcgga 180 tgtggtacta
cggacaagac ctggggtcaa tccgacccag cggcaagagc taaggccaaa 240
gttgatgctg ctttcgaaat tatggataag ctgagcattg attactactg cttccatgat
300 agagaccttt ctccagaata tggctccttg aaagcgacca atgatcaact
ggacattgtt 360 actgattaca tcaaggagaa gcagggcgat aaattcaagt
gtttatgggg cactgctaaa 420 tgctttgatc accccaggtt catgcacggt
gcaggaactt ctcctagtgc cgatgttttc 480 gctttttctg ctgcgcaaat
aaagaaagca ttagaatcta ccgtcaagtt gggcggtaat 540 ggttatgtct
tttggggtgg tagagaaggt tacgagaccc tgctgaatac taacatgggc 600
ttagaactgg acaacatggc taggctaatg aagatggccg tagaatacgg taggtctatt
660 ggattcaaag gtgacttcta catcgagcct aaacccaagg aacctactaa
gcaccagtac 720 gacttcgaca ctgctaccgt attaggtttt ttaaggaagt
acgggttgga taaagacttc 780 aagatgaaca tcgaagccaa tcacgccaca
ctagcacaac acacattcca gcatgagtta 840 cgtgtggcta gggataacgg
tgtattcggt tctattgatg ctaaccaagg tgacgtattg 900 ttaggatggg
acacggatca attccccaca aacatttatg atactactat gtgtatgtat 960
gaggtcatta aagccggtgg tttcacaaat ggcggcctga actttgatgc gaaagctcgt
1020 aggggttcat tcacgcctga agatattttc tatagttaca ttgctggtat
ggatgctttc 1080 gcgttagggt ttagagcagc tcttaaattg attgaagacg
gtagaattga caagtttgtg 1140 gctgacaggt atgcctcttg gaataccggt
attggtgcag atattattgc cggaaaagcc 1200 gattttgcat cattggaaaa
atatgctttg gaaaaaggtg aagttaccgc gtcattgtct 1260 tctggtagac
aagagatgct ggaatctatt gtcaacaacg tattgtttag tttgtaataa 1320
<210> SEQ ID NO 4 <211> LENGTH: 438 <212> TYPE:
PRT <213> ORGANISM: Ruminococcus flavefaciens <400>
SEQUENCE: 4 Met Glu Phe Phe Ser Asn Ile Gly Lys Ile Gln Tyr Gln Gly
Pro Lys 1 5 10 15 Ser Thr Asp Pro Leu Ser Phe Lys Tyr Tyr Asn Pro
Glu Glu Val Ile 20 25 30 Asn Gly Lys Thr Met Arg Glu His Leu Lys
Phe Ala Leu Ser Trp Trp 35 40 45 His Thr Met Gly Gly Asp Gly Thr
Asp Met Phe Gly Cys Gly Thr Thr 50 55 60 Asp Lys Thr Trp Gly Gln
Ser Asp Pro Ala Ala Arg Ala Lys Ala Lys 65 70 75 80 Val Asp Ala Ala
Phe Glu Ile Met Asp Lys Leu Ser Ile Asp Tyr Tyr 85 90 95 Cys Phe
His Asp Arg Asp Leu Ser Pro Glu Tyr Gly Ser Leu Lys Ala 100 105 110
Thr Asn Asp Gln Leu Asp Ile Val Thr Asp Tyr Ile Lys Glu Lys Gln 115
120 125 Gly Asp Lys Phe Lys Cys Leu Trp Gly Thr Ala Lys Cys Phe Asp
His 130 135 140 Pro Arg Phe Met His Gly Ala Gly Thr Ser Pro Ser Ala
Asp Val Phe 145 150 155 160 Ala Phe Ser Ala Ala Gln Ile Lys Lys Ala
Leu Glu Ser Thr Val Lys 165 170 175 Leu Gly Gly Asn Gly Tyr Val Phe
Trp Gly Gly Arg Glu Gly Tyr Glu 180 185 190 Thr Leu Leu Asn Thr Asn
Met Gly Leu Glu Leu Asp Asn Met Ala Arg
195 200 205 Leu Met Lys Met Ala Val Glu Tyr Gly Arg Ser Ile Gly Phe
Lys Gly 210 215 220 Asp Phe Tyr Ile Glu Pro Lys Pro Lys Glu Pro Thr
Lys His Gln Tyr 225 230 235 240 Asp Phe Asp Thr Ala Thr Val Leu Gly
Phe Leu Arg Lys Tyr Gly Leu 245 250 255 Asp Lys Asp Phe Lys Met Asn
Ile Glu Ala Asn His Ala Thr Leu Ala 260 265 270 Gln His Thr Phe Gln
His Glu Leu Arg Val Ala Arg Asp Asn Gly Val 275 280 285 Phe Gly Ser
Ile Asp Ala Asn Gln Gly Asp Val Leu Leu Gly Trp Asp 290 295 300 Thr
Asp Gln Phe Pro Thr Asn Ile Tyr Asp Thr Thr Met Cys Met Tyr 305 310
315 320 Glu Val Ile Lys Ala Gly Gly Phe Thr Asn Gly Gly Leu Asn Phe
Asp 325 330 335 Ala Lys Ala Arg Arg Gly Ser Phe Thr Pro Glu Asp Ile
Phe Tyr Ser 340 345 350 Tyr Ile Ala Gly Met Asp Ala Phe Ala Leu Gly
Phe Arg Ala Ala Leu 355 360 365 Lys Leu Ile Glu Asp Gly Arg Ile Asp
Lys Phe Val Ala Asp Arg Tyr 370 375 380 Ala Ser Trp Asn Thr Gly Ile
Gly Ala Asp Ile Ile Ala Gly Lys Ala 385 390 395 400 Asp Phe Ala Ser
Leu Glu Lys Tyr Ala Leu Glu Lys Gly Glu Val Thr 405 410 415 Ala Ser
Leu Ser Ser Gly Arg Gln Glu Met Leu Glu Ser Ile Val Asn 420 425 430
Asn Val Leu Phe Ser Leu 435 <210> SEQ ID NO 5 <211>
LENGTH: 14 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic DNA polynucleotide <400> SEQUENCE: 5 ggatcccaaa
caaa 14 <210> SEQ ID NO 6 <400> SEQUENCE: 6 000
<210> SEQ ID NO 7 <211> LENGTH: 88 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic DNA polynucleotide
<400> SEQUENCE: 7 gagctcacgg atccgtcata tgctagatct ctgaattctt
actagttcga cgtctaccta 60 ggcagtcgac acgcggccgc ttctcgag 88
<210> SEQ ID NO 8 <211> LENGTH: 1317 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic DNA polynucleotide codon
optimized for expression of xylose isomerase in yeast <400>
SEQUENCE: 8 atggtgaacg gtttgaccaa catcccacca gtcaaattcg aaggtagaga
ctccaaaaaa 60 gcattgtctt ttaaatatta taatcctgat gaaatgatcc
aaggtaagaa aatgaaggat 120 tatttgaaat tcgctatgtc ctattggcac
actttgtgtg gcgatggtac tgatccattc 180 ggatcatcta ctattgacag
ggactacagt ggccaaaccc caatggaaaa agctaagacc 240 aaagctgatg
ttgctttcgc actgatgcaa attctgggca tcgagtactt ctgcttccat 300
gatttggaca ttgctccaac aggtaactcc ttgaaagagt tgaagaataa tctgatcgag
360 attactgatt acatcaaggg gttgatggac aaaaccggca tcaagttgtt
atggggcact 420 gctaactgct ttagtcaccc caggtacatg aacggtgcag
gaacttctcc tcaagccgat 480 attttcgctt gtgctgctgc gcaaataaag
aacgctatag atgcgaccat caagttgggc 540 ggtacgggtt atgtcttttg
gggtggtaga gaaggttacg agaccctgaa tactaacatg 600 gaaatagaac
tggacaacat ggctaagcta atgcacatgg ccgtagatta cgctaggtct 660
aaaggattca ccggtgactt ctacatcgag cctaaaccca aggaacctac taagcaccag
720 tacgacttcg acgttgctac cgtagtaggt tttttaagga agtacgggtt
ggataaagac 780 ttcaagatga acatcgaagc caatcacgcc acactagcag
gccacacatt ccagcatgag 840 ttaaatgtgg ctagggtaaa caatgtattc
ggttctattg atgctaacca aggtgaccta 900 ttgttaggat gggacacgga
tcaattcccc acaaacgttt atgatactac tctttgtatg 960 ctggaggtca
ttaaagccgg tggtttcaca aatggcggcc tgaactttga tgcgaaagtt 1020
cgtagggctt catacacgat ggaagatatt atcttggctt acatttctgg tatggatact
1080 ttcgcgttag ggttaaaaat agctaataaa atcattgaag acggtagaat
tgacgagttt 1140 gtgtctagga ggtatgcctc ttacaagacc ggtattggtg
cagatattat tgccggaaga 1200 accaatttgg aagaattgga aaaatatgct
ttggaacttc ctccagttga accgcatcct 1260 ggtaaacaag agtatctgga
agctgttttc aacaacgtaa tgtttacagt ttaataa 1317 <210> SEQ ID NO
9 <211> LENGTH: 437 <212> TYPE: PRT <213>
ORGANISM: Ruminococcus flavefaciens <400> SEQUENCE: 9 Met Val
Asn Gly Leu Thr Asn Ile Pro Pro Val Lys Phe Glu Gly Arg 1 5 10 15
Asp Ser Lys Lys Ala Leu Ser Phe Lys Tyr Tyr Asn Pro Asp Glu Met 20
25 30 Ile Gln Gly Lys Lys Met Lys Asp Tyr Leu Lys Phe Ala Met Ser
Tyr 35 40 45 Trp His Thr Leu Cys Gly Asp Gly Thr Asp Pro Phe Gly
Ser Ser Thr 50 55 60 Ile Asp Arg Asp Tyr Ser Gly Gln Thr Pro Met
Glu Lys Ala Lys Thr 65 70 75 80 Lys Ala Asp Val Ala Phe Ala Leu Met
Gln Ile Leu Gly Ile Glu Tyr 85 90 95 Phe Cys Phe His Asp Leu Asp
Ile Ala Pro Thr Gly Asn Ser Leu Lys 100 105 110 Glu Leu Lys Asn Asn
Leu Ile Glu Ile Thr Asp Tyr Ile Lys Gly Leu 115 120 125 Met Asp Lys
Thr Gly Ile Lys Leu Leu Trp Gly Thr Ala Asn Cys Phe 130 135 140 Ser
His Pro Arg Tyr Met Asn Gly Ala Gly Thr Ser Pro Gln Ala Asp 145 150
155 160 Ile Phe Ala Cys Ala Ala Ala Gln Ile Lys Asn Ala Ile Asp Ala
Thr 165 170 175 Ile Lys Leu Gly Gly Thr Gly Tyr Val Phe Trp Gly Gly
Arg Glu Gly 180 185 190 Tyr Glu Thr Leu Asn Thr Asn Met Glu Ile Glu
Leu Asp Asn Met Ala 195 200 205 Lys Leu Met His Met Ala Val Asp Tyr
Ala Arg Ser Lys Gly Phe Thr 210 215 220 Gly Asp Phe Tyr Ile Glu Pro
Lys Pro Lys Glu Pro Thr Lys His Gln 225 230 235 240 Tyr Asp Phe Asp
Val Ala Thr Val Val Gly Phe Leu Arg Lys Tyr Gly 245 250 255 Leu Asp
Lys Asp Phe Lys Met Asn Ile Glu Ala Asn His Ala Thr Leu 260 265 270
Ala Gly His Thr Phe Gln His Glu Leu Asn Val Ala Arg Val Asn Asn 275
280 285 Val Phe Gly Ser Ile Asp Ala Asn Gln Gly Asp Leu Leu Leu Gly
Trp 290 295 300 Asp Thr Asp Gln Phe Pro Thr Asn Val Tyr Asp Thr Thr
Leu Cys Met 305 310 315 320 Leu Glu Val Ile Lys Ala Gly Gly Phe Thr
Asn Gly Gly Leu Asn Phe 325 330 335 Asp Ala Lys Val Arg Arg Ala Ser
Tyr Thr Met Glu Asp Ile Ile Leu 340 345 350 Ala Tyr Ile Ser Gly Met
Asp Thr Phe Ala Leu Gly Leu Lys Ile Ala 355 360 365 Asn Lys Ile Ile
Glu Asp Gly Arg Ile Asp Glu Phe Val Ser Arg Arg 370 375 380 Tyr Ala
Ser Tyr Lys Thr Gly Ile Gly Ala Asp Ile Ile Ala Gly Arg 385 390 395
400 Thr Asn Leu Glu Glu Leu Glu Lys Tyr Ala Leu Glu Leu Pro Pro Val
405 410 415 Glu Pro His Pro Gly Lys Gln Glu Tyr Leu Glu Ala Val Phe
Asn Asn 420 425 430 Val Met Phe Thr Val 435 <210> SEQ ID NO
10 <211> LENGTH: 1317 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic DNA polynucleotide codon optimized for
expression of xylose isomerase in yeast <400> SEQUENCE: 10
atgagggaac atttcttgga aatcaataaa atcaaattcg aaggtggaga ctccacaaat
60 cctttggctt ttaaatatta tgatgctaat agaatagtcg ccggtaagaa
aatgaaggat 120 catttgagat tcgctctatc ctattggcac actttgactg
gcaatggtac tgatccattc 180 ggacaaccta ctatggaaag ggactacaat
agccttgacg gaattgaact atctaaggcc 240 agagttgatg ctgctttcga
actgatgact aagctgggca tcgagttctt ctgcttccat 300 gatttggaca
ttgctccaga aggtaactcc ttgcaagaga aattggataa tctggacacg 360
attcttgaaa gaatcgagga taagatgaaa gaaaccggca tcaagtgttt atggggcact
420 actaacgcct ttagtcaccc caggttcatg cacggtgcag caacttctcc
taatgccgat 480 gttttcgctt ttgctgctgc gcaagtaaag aaagcattag
aaattaccca caggttgaga 540 ggtgagaatt atgtcttttg gggtggtaga
gaaggttacg agaccctgct gaatactgac 600 attgccttag aaaatgacaa
cttggctaag tttttgaaga tggccaaaga ttacgctagg 660 aatattggat
tcgaaggtca attcttgatc gagcctaaac ccaaggaacc tactaagcac 720
cagtacgact tcgacactat gaccgtatta ggttttttaa ggaagtacaa tttgatagat
780 gacttcaagt tgaacatcga agccaatcac gccacactag caggccacac
attccagcat 840 gagttagcta tggctaggat aaacggtgta ttgggttctg
ttgatgctaa ccaaggtgac 900 ctattgttag gatgggacac ggatcaattc
cccacaaaca tttatgatgc tactctttct 960 atgtatgagg tcttgaaaaa
cggtggtatc gcacctggcg gcctgaactt tgatgcgaaa 1020 gttcgtaggg
gttcattcaa gcctgacgat cttttcattg cttacattgt tggtatggat 1080
actttcgcga aagggttact tgtagctgat aaattgctta ctgacggtgt acttgaaaat
1140 tttgtgacta aaaggtatga atcttacact gctggtattg gtaaaaagat
cattgaagat 1200 gctacctctt ttgaagaatt ggcagaatat gctttgaaac
atgataaaat tgtcttggaa 1260 tctggtagac aagagatgct ggaagatatt
gtcaacagat atatttataa ataataa 1317 <210> SEQ ID NO 11
<211> LENGTH: 437 <212> TYPE: PRT <213> ORGANISM:
Alkaliphilus metalliredigens <400> SEQUENCE: 11 Met Arg Glu
His Phe Leu Glu Ile Asn Lys Ile Lys Phe Glu Gly Gly 1 5 10 15 Asp
Ser Thr Asn Pro Leu Ala Phe Lys Tyr Tyr Asp Ala Asn Arg Ile 20 25
30 Val Ala Gly Lys Lys Met Lys Asp His Leu Arg Phe Ala Leu Ser Tyr
35 40 45 Trp His Thr Leu Thr Gly Asn Gly Thr Asp Pro Phe Gly Gln
Pro Thr 50 55 60 Met Glu Arg Asp Tyr Asn Ser Leu Asp Gly Ile Glu
Leu Ser Lys Ala 65 70 75 80 Arg Val Asp Ala Ala Phe Glu Leu Met Thr
Lys Leu Gly Ile Glu Phe 85 90 95 Phe Cys Phe His Asp Leu Asp Ile
Ala Pro Glu Gly Asn Ser Leu Gln 100 105 110 Glu Lys Leu Asp Asn Leu
Asp Thr Ile Leu Glu Arg Ile Glu Asp Lys 115 120 125 Met Lys Glu Thr
Gly Ile Lys Cys Leu Trp Gly Thr Thr Asn Ala Phe 130 135 140 Ser His
Pro Arg Phe Met His Gly Ala Ala Thr Ser Pro Asn Ala Asp 145 150 155
160 Val Phe Ala Phe Ala Ala Ala Gln Val Lys Lys Ala Leu Glu Ile Thr
165 170 175 His Arg Leu Arg Gly Glu Asn Tyr Val Phe Trp Gly Gly Arg
Glu Gly 180 185 190 Tyr Glu Thr Leu Leu Asn Thr Asp Ile Ala Leu Glu
Asn Asp Asn Leu 195 200 205 Ala Lys Phe Leu Lys Met Ala Lys Asp Tyr
Ala Arg Asn Ile Gly Phe 210 215 220 Glu Gly Gln Phe Leu Ile Glu Pro
Lys Pro Lys Glu Pro Thr Lys His 225 230 235 240 Gln Tyr Asp Phe Asp
Thr Met Thr Val Leu Gly Phe Leu Arg Lys Tyr 245 250 255 Asn Leu Ile
Asp Asp Phe Lys Leu Asn Ile Glu Ala Asn His Ala Thr 260 265 270 Leu
Ala Gly His Thr Phe Gln His Glu Leu Ala Met Ala Arg Ile Asn 275 280
285 Gly Val Leu Gly Ser Val Asp Ala Asn Gln Gly Asp Leu Leu Leu Gly
290 295 300 Trp Asp Thr Asp Gln Phe Pro Thr Asn Ile Tyr Asp Ala Thr
Leu Ser 305 310 315 320 Met Tyr Glu Val Leu Lys Asn Gly Gly Ile Ala
Pro Gly Gly Leu Asn 325 330 335 Phe Asp Ala Lys Val Arg Arg Gly Ser
Phe Lys Pro Asp Asp Leu Phe 340 345 350 Ile Ala Tyr Ile Val Gly Met
Asp Thr Phe Ala Lys Gly Leu Leu Val 355 360 365 Ala Asp Lys Leu Leu
Thr Asp Gly Val Leu Glu Asn Phe Val Thr Lys 370 375 380 Arg Tyr Glu
Ser Tyr Thr Ala Gly Ile Gly Lys Lys Ile Ile Glu Asp 385 390 395 400
Ala Thr Ser Phe Glu Glu Leu Ala Glu Tyr Ala Leu Lys His Asp Lys 405
410 415 Ile Val Leu Glu Ser Gly Arg Gln Glu Met Leu Glu Asp Ile Val
Asn 420 425 430 Arg Tyr Ile Tyr Lys 435 <210> SEQ ID NO 12
<211> LENGTH: 1314 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic DNA polynucleotide codon optimized for
expression of xylose isomerase in yeast <400> SEQUENCE: 12
atggaatttt tcaaaggcat tgataaagtc aaatacgaag gtgtaaaaac caataatctt
60 ttggcttttg cacattataa tcctgaagaa gtaatcttgg gtaagaaaat
gaaggatcat 120 ttgaaattcg ctatgtccta ttggcacact ttgactggcg
aaggtactga tccattcgga 180 aatgctacta tggacaggga atggaatgaa
tataccccaa tggaaaaagc taaggccaga 240 gttaaagctg gtttcgaatt
tatggaaaag ctgggcttgg agtacttctg cttccatgat 300 aaagacattg
ctccagaagc tgaaaccttg gaagagtacc ataggaatct ggacgagatt 360
gttgatttga tcgaggagga gatgaaaaga accggcatca agttgttatg gggcacttct
420 aacatgttta gtcaccccag gttcatgcac ggtgcagcaa cttcttgtaa
tgccgatgtt 480 ttcgcttatg ctgctgcgca aacaaagaaa gcattagaaa
ttaccaaaag gttgaacggt 540 acgggttatg tcttttgggg tggtagagaa
ggttacgaga ccctgctgaa tactgacatt 600 ggcttagaac tggacaactt
ggctaggttt ttgcagatgg ccgtagatta cgctaagaaa 660 attggattcg
aaggtcaatt cttcatcgag cctaaaccca aggaacctac taagcaccag 720
tacgacttcg acactactac cgtattagaa tttttaagga agtacaattt ggataaatac
780 ttcaagatga acatcgaagc caatcacgcc acactagcag gccacacatt
ccagcatgag 840 ttatgtacgg ctaggataaa cggtgtattc ggttctattg
atgctaacca aggtgacatg 900 ttgttaggat gggacacgga tcaattcccc
acaaacgttt atgatgctgt tcttgctatg 960 tatgagacct tgttagccgg
tggtttcaaa gaaggcggcc tgaactttga tgcgaaagtt 1020 cgtaggggtt
cattcgagcc taaagatttg ttctatgctt acatttctgg tatggatact 1080
ttcgcgaaag ggttaaaagt agctgctaaa ttgattgaag acggtacatt tgaaaagatt
1140 aaggttgaaa ggtattcctc ttacacgacc ggtattggta aacagatcgt
taacggagaa 1200 gtcggttttg aagaattgtc aaaatatgct ttgactaatg
gtgtaaaaaa aaattcatct 1260 ggtagacaag agatgctgga aaatattttg
aacagatata tttatgaata ataa 1314 <210> SEQ ID NO 13
<211> LENGTH: 436 <212> TYPE: PRT <213> ORGANISM:
Fusobacterium mortiferum <400> SEQUENCE: 13 Met Glu Phe Phe
Lys Gly Ile Asp Lys Val Lys Tyr Glu Gly Val Lys 1 5 10 15 Thr Asn
Asn Leu Leu Ala Phe Ala His Tyr Asn Pro Glu Glu Val Ile 20 25 30
Leu Gly Lys Lys Met Lys Asp His Leu Lys Phe Ala Met Ser Tyr Trp 35
40 45 His Thr Leu Thr Gly Glu Gly Thr Asp Pro Phe Gly Asn Ala Thr
Met 50 55 60 Asp Arg Glu Trp Asn Glu Tyr Thr Pro Met Glu Lys Ala
Lys Ala Arg 65 70 75 80 Val Lys Ala Gly Phe Glu Phe Met Glu Lys Leu
Gly Leu Glu Tyr Phe 85 90 95 Cys Phe His Asp Lys Asp Ile Ala Pro
Glu Ala Glu Thr Leu Glu Glu 100 105 110 Tyr His Arg Asn Leu Asp Glu
Ile Val Asp Leu Ile Glu Glu Glu Met 115 120 125 Lys Arg Thr Gly Ile
Lys Leu Leu Trp Gly Thr Ser Asn Met Phe Ser 130 135 140 His Pro Arg
Phe Met His Gly Ala Ala Thr Ser Cys Asn Ala Asp Val 145 150 155 160
Phe Ala Tyr Ala Ala Ala Gln Thr Lys Lys Ala Leu Glu Ile Thr Lys 165
170 175 Arg Leu Asn Gly Thr Gly Tyr Val Phe Trp Gly Gly Arg Glu Gly
Tyr 180 185 190 Glu Thr Leu Leu Asn Thr Asp Ile Gly Leu Glu Leu Asp
Asn Leu Ala 195 200 205 Arg Phe Leu Gln Met Ala Val Asp Tyr Ala Lys
Lys Ile Gly Phe Glu 210 215 220 Gly Gln Phe Phe Ile Glu Pro Lys Pro
Lys Glu Pro Thr Lys His Gln 225 230 235 240 Tyr Asp Phe Asp Thr Thr
Thr Val Leu Glu Phe Leu Arg Lys Tyr Asn 245 250 255 Leu Asp Lys Tyr
Phe Lys Met Asn Ile Glu Ala Asn His Ala Thr Leu 260 265 270 Ala Gly
His Thr Phe Gln His Glu Leu Cys Thr Ala Arg Ile Asn Gly 275 280 285
Val Phe Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu Gly Trp 290
295 300 Asp Thr Asp Gln Phe Pro Thr Asn Val Tyr Asp Ala Val Leu Ala
Met 305 310 315 320 Tyr Glu Thr Leu Leu Ala Gly Gly Phe Lys Glu Gly
Gly Leu Asn Phe 325 330 335
Asp Ala Lys Val Arg Arg Gly Ser Phe Glu Pro Lys Asp Leu Phe Tyr 340
345 350 Ala Tyr Ile Ser Gly Met Asp Thr Phe Ala Lys Gly Leu Lys Val
Ala 355 360 365 Ala Lys Leu Ile Glu Asp Gly Thr Phe Glu Lys Ile Lys
Val Glu Arg 370 375 380 Tyr Ser Ser Tyr Thr Thr Gly Ile Gly Lys Gln
Ile Val Asn Gly Glu 385 390 395 400 Val Gly Phe Glu Glu Leu Ser Lys
Tyr Ala Leu Thr Asn Gly Val Lys 405 410 415 Lys Asn Ser Ser Gly Arg
Gln Glu Met Leu Glu Asn Ile Leu Asn Arg 420 425 430 Tyr Ile Tyr Glu
435 <210> SEQ ID NO 14 <211> LENGTH: 1323 <212>
TYPE: DNA <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic DNA
polynucleotide codon optimized for expression of xylose isomerase
in yeast <400> SEQUENCE: 14 atgagtgaag ttttctccgg catctcaaat
atcaaattcg aaggttcagg ctccgataat 60 cctttggctt ttaaatatta
tgatcctaaa gcagtaatcg gcggtaagac tatggaagag 120 catttgagat
tcgctgtagc ctattggcac acttttgctg cccctggtgc tgatatgttc 180
ggagcaggtt cttatgtcag gccctggaat accatgtccg acccattgga aatagctaag
240 tacaaagttg aagctaattt cgaatttatt gaaaagctgg gcgccccgtt
cttcgccttc 300 catgatagag acattgctcc agaaggtgac accttggcag
agaccaataa gaatctggac 360 acgattgttt ctgtcatcaa ggataggatg
aaatcatccc ccgtcaagtt gttatggggc 420 actactaacg cctttggtaa
ccccaggttc atgcacggtg catcaacttc tcctaatgcc 480 gatattttcg
cttatgctgc tgcgcaagta aagaaagcta tggaaattac caaagagttg 540
ggcggtgaga attatgtctt ttggggtggt agagaaggtt acgagaccct gctgaatact
600 gacatgaaat tagaactgga caacttggct aggtttttga agatggccgt
agattacgct 660 aaggaaattg gattcgacgg tcaattcttg atcgagccta
aacccaagga acctactaag 720 caccagtacg acttcgacac tgctaccgta
ataggttttt taaagacgta cgggttggac 780 ccctacttca agatgaacat
cgaagccaat cacgccacac tagcaggcca cacattccag 840 catgagttag
ctatgtgtag gataaacgat atgttgggtt ctattgatgc taaccaaggt 900
gacgtaatgt taggatggga cacggatcaa ttccccacaa acctttatga tgctactctt
960 gctatggtgg aggtcttgaa agccggtggt ttgaaaaaag gcggcctgaa
ctttgattct 1020 aaagttcgta ggggttcatt cgagccttcc gatcttttct
atggtcacat tgctggtatg 1080 gatactttcg cgaaagggtt aattatagct
aataaaatcg ttgaagacgg taaatttgat 1140 gcttttgtgg ctgacaggta
ttcctcttac acgaacggta ttggtaaaga tattgttgaa 1200 ggaaaagtcg
gttttaaaga attggaacaa tatgctttga ctgctaaaat tcaaaataaa 1260
tctggtagac aagagatgct ggaagctctt ttgaaccaat atattttgga aactaaataa
1320 taa 1323 <210> SEQ ID NO 15 <211> LENGTH: 439
<212> TYPE: PRT <213> ORGANISM: Clostridium
cellulolyticum <400> SEQUENCE: 15 Met Ser Glu Val Phe Ser Gly
Ile Ser Asn Ile Lys Phe Glu Gly Ser 1 5 10 15 Gly Ser Asp Asn Pro
Leu Ala Phe Lys Tyr Tyr Asp Pro Lys Ala Val 20 25 30 Ile Gly Gly
Lys Thr Met Glu Glu His Leu Arg Phe Ala Val Ala Tyr 35 40 45 Trp
His Thr Phe Ala Ala Pro Gly Ala Asp Met Phe Gly Ala Gly Ser 50 55
60 Tyr Val Arg Pro Trp Asn Thr Met Ser Asp Pro Leu Glu Ile Ala Lys
65 70 75 80 Tyr Lys Val Glu Ala Asn Phe Glu Phe Ile Glu Lys Leu Gly
Ala Pro 85 90 95 Phe Phe Ala Phe His Asp Arg Asp Ile Ala Pro Glu
Gly Asp Thr Leu 100 105 110 Ala Glu Thr Asn Lys Asn Leu Asp Thr Ile
Val Ser Val Ile Lys Asp 115 120 125 Arg Met Lys Ser Ser Pro Val Lys
Leu Leu Trp Gly Thr Thr Asn Ala 130 135 140 Phe Gly Asn Pro Arg Phe
Met His Gly Ala Ser Thr Ser Pro Asn Ala 145 150 155 160 Asp Ile Phe
Ala Tyr Ala Ala Ala Gln Val Lys Lys Ala Met Glu Ile 165 170 175 Thr
Lys Glu Leu Gly Gly Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu 180 185
190 Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Lys Leu Glu Leu Asp Asn
195 200 205 Leu Ala Arg Phe Leu Lys Met Ala Val Asp Tyr Ala Lys Glu
Ile Gly 210 215 220 Phe Asp Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys
Glu Pro Thr Lys 225 230 235 240 His Gln Tyr Asp Phe Asp Thr Ala Thr
Val Ile Gly Phe Leu Lys Thr 245 250 255 Tyr Gly Leu Asp Pro Tyr Phe
Lys Met Asn Ile Glu Ala Asn His Ala 260 265 270 Thr Leu Ala Gly His
Thr Phe Gln His Glu Leu Ala Met Cys Arg Ile 275 280 285 Asn Asp Met
Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Val Met Leu 290 295 300 Gly
Trp Asp Thr Asp Gln Phe Pro Thr Asn Leu Tyr Asp Ala Thr Leu 305 310
315 320 Ala Met Val Glu Val Leu Lys Ala Gly Gly Leu Lys Lys Gly Gly
Leu 325 330 335 Asn Phe Asp Ser Lys Val Arg Arg Gly Ser Phe Glu Pro
Ser Asp Leu 340 345 350 Phe Tyr Gly His Ile Ala Gly Met Asp Thr Phe
Ala Lys Gly Leu Ile 355 360 365 Ile Ala Asn Lys Ile Val Glu Asp Gly
Lys Phe Asp Ala Phe Val Ala 370 375 380 Asp Arg Tyr Ser Ser Tyr Thr
Asn Gly Ile Gly Lys Asp Ile Val Glu 385 390 395 400 Gly Lys Val Gly
Phe Lys Glu Leu Glu Gln Tyr Ala Leu Thr Ala Lys 405 410 415 Ile Gln
Asn Lys Ser Gly Arg Gln Glu Met Leu Glu Ala Leu Leu Asn 420 425 430
Gln Tyr Ile Leu Glu Thr Lys 435 <210> SEQ ID NO 16
<211> LENGTH: 1320 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic DNA polynucleotide codon optimized for
expression of xylose isomerase in yeast <400> SEQUENCE: 16
atgaagaact atttccccaa cgtcccagaa gtcaaatacg aaggtccaaa ctccacaaat
60 cctttcgctt ttaaatatta tgatgctaat aaagtagtcg ccggtaagac
catgaaggag 120 cattgtagat tcgctctatc ctggtggcac actttgtgtg
ccggtggtgc tgatccattc 180 ggagtaacta ctatggacag gacctacggt
aacattaccg acccaatgga actagctaag 240 gccaaagttg atgctggttt
cgaactgatg actaagctgg gcatcgagtt cttctgcttc 300 catgatgccg
acattgctcc agaaggtgac accttcgaag agtccaagaa gaatctgttc 360
gagattgttg attacatcaa ggagaagatg gaccaaaccg gcatcaagtt gttatggggc
420 actgctaaca actttagtca ccccaggttc atgcacggtg catcaacttc
ttgtaatgcc 480 gatgttttcg cttatgctgc tgcgaaaata aagaacgctt
tagatgcgac catcaagttg 540 ggcggtaagg gttatgtctt ttggggtggt
agagaaggtt acgagaccct gctgaatact 600 gacctgggct tagaactgga
caacatggct aggctaatga agatggccgt agaatacggt 660 agggctaatg
gattcgacgg tgacttctac atcgagccta aacccaagga acctactaag 720
caccagtacg acttcgacac tgctaccgta ttagcttttt taaggaagta cgggttggaa
780 aaagacttca agatgaacat cgaagccaat cacgccacac tagcaggcca
cacattcgag 840 catgagttag ctatggctag ggtaaacggt gcattcggtt
ctgttgatgc taaccaaggt 900 gacccaaact taggatggga cacggatcaa
ttccccacag acgttcattc tgctactctt 960 gctatgctgg aggtcttgaa
agccggtggt ttcacaaatg gcggcctgaa ctttgatgcg 1020 aaagttcgta
ggggttcatt cgagtttgac gatattgcct atggttacat tgctggtatg 1080
gatactttcg cgttagggtt aattaaagct gctgaaatca ttgatgacgg tagaattgcc
1140 aagtttgtgg atgacaggta tgcctcttac aagaccggta ttggtaaagc
gatcgttgac 1200 ggaactacct ctttggaaga attggaacaa tacgtgttga
ctcattctga acctgtcatg 1260 caatctggta gacaagaggt tctggaaact
attgtcaaca acatattgtt tagataataa 1320 <210> SEQ ID NO 17
<211> LENGTH: 438 <212> TYPE: PRT <213> ORGANISM:
Clostridium phytofermentans <400> SEQUENCE: 17 Met Lys Asn
Tyr Phe Pro Asn Val Pro Glu Val Lys Tyr Glu Gly Pro 1 5 10 15 Asn
Ser Thr Asn Pro Phe Ala Phe Lys Tyr Tyr Asp Ala Asn Lys Val 20 25
30 Val Ala Gly Lys Thr Met Lys Glu His Cys Arg Phe Ala Leu Ser Trp
35 40 45 Trp His Thr Leu Cys Ala Gly Gly Ala Asp Pro Phe Gly Val
Thr Thr 50 55 60 Met Asp Arg Thr Tyr Gly Asn Ile Thr Asp Pro Met
Glu Leu Ala Lys 65 70 75 80
Ala Lys Val Asp Ala Gly Phe Glu Leu Met Thr Lys Leu Gly Ile Glu 85
90 95 Phe Phe Cys Phe His Asp Ala Asp Ile Ala Pro Glu Gly Asp Thr
Phe 100 105 110 Glu Glu Ser Lys Lys Asn Leu Phe Glu Ile Val Asp Tyr
Ile Lys Glu 115 120 125 Lys Met Asp Gln Thr Gly Ile Lys Leu Leu Trp
Gly Thr Ala Asn Asn 130 135 140 Phe Ser His Pro Arg Phe Met His Gly
Ala Ser Thr Ser Cys Asn Ala 145 150 155 160 Asp Val Phe Ala Tyr Ala
Ala Ala Lys Ile Lys Asn Ala Leu Asp Ala 165 170 175 Thr Ile Lys Leu
Gly Gly Lys Gly Tyr Val Phe Trp Gly Gly Arg Glu 180 185 190 Gly Tyr
Glu Thr Leu Leu Asn Thr Asp Leu Gly Leu Glu Leu Asp Asn 195 200 205
Met Ala Arg Leu Met Lys Met Ala Val Glu Tyr Gly Arg Ala Asn Gly 210
215 220 Phe Asp Gly Asp Phe Tyr Ile Glu Pro Lys Pro Lys Glu Pro Thr
Lys 225 230 235 240 His Gln Tyr Asp Phe Asp Thr Ala Thr Val Leu Ala
Phe Leu Arg Lys 245 250 255 Tyr Gly Leu Glu Lys Asp Phe Lys Met Asn
Ile Glu Ala Asn His Ala 260 265 270 Thr Leu Ala Gly His Thr Phe Glu
His Glu Leu Ala Met Ala Arg Val 275 280 285 Asn Gly Ala Phe Gly Ser
Val Asp Ala Asn Gln Gly Asp Pro Asn Leu 290 295 300 Gly Trp Asp Thr
Asp Gln Phe Pro Thr Asp Val His Ser Ala Thr Leu 305 310 315 320 Ala
Met Leu Glu Val Leu Lys Ala Gly Gly Phe Thr Asn Gly Gly Leu 325 330
335 Asn Phe Asp Ala Lys Val Arg Arg Gly Ser Phe Glu Phe Asp Asp Ile
340 345 350 Ala Tyr Gly Tyr Ile Ala Gly Met Asp Thr Phe Ala Leu Gly
Leu Ile 355 360 365 Lys Ala Ala Glu Ile Ile Asp Asp Gly Arg Ile Ala
Lys Phe Val Asp 370 375 380 Asp Arg Tyr Ala Ser Tyr Lys Thr Gly Ile
Gly Lys Ala Ile Val Asp 385 390 395 400 Gly Thr Thr Ser Leu Glu Glu
Leu Glu Gln Tyr Val Leu Thr His Ser 405 410 415 Glu Pro Val Met Gln
Ser Gly Arg Gln Glu Val Leu Glu Thr Ile Val 420 425 430 Asn Asn Ile
Leu Phe Arg 435 <210> SEQ ID NO 18 <211> LENGTH: 1317
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic DNA
polynucleotide <400> SEQUENCE: 18 atggaatttt tctccaacat
cggaaaaatc caataccaag gtccaaaatc cacagatcct 60 ttgtctttta
aatattataa tcctgaagaa gtaatcaacg gtaagaccat gagggagcat 120
ttgaaattcg ctctatcatg gtggcacaca atgggtggcg atggtactga tatgttcgga
180 tgtggtacta cggacaagac ctggggtcaa tccgacccag cggcaagagc
taaggccaaa 240 gttgatgctg ctttcgaaat tatggataag ctgagcattg
attactactg cttccatgat 300 agagaccttt ctccagaata tggctccttg
aaagcgacca atgatcaact ggacattgtt 360 actgattaca tcaaggagaa
gcagggcgat aaattcaagt gtttatgggg cactgctaaa 420 tgctttgatc
accccaggtt catgcacggt gcaggaactt ctcctagtgc cgatgttttc 480
gctttttctg ctgcgcaaat aaagaaagca ttagaatcta ccgtcaagtt gggcggtaat
540 ggttatgtct tttggggtgg tagagaaggt tacgagaccc tgctgaatac
taacatgggc 600 ttagaactgg acaacatggc taggctaatg aagatggccg
tagaatacgg taggtctatt 660 ggattcaaag gtgacttcta catcgagcct
aaacccaagg aacctactaa gcaccagtac 720 gacttcgaca ctgctaccgt
attaggtttt ttaaggaagt acgggttgga taaagacttc 780 aaaatgaaca
tcgaagccaa tcacgccaca ctagcacaac acacattcca gcatgagtta 840
cgtgtggcta gggataacgg tgtattcggt tctattgatg ctaaccaagg tgacgtattg
900 ttaggatggg acacggatca attccccaca aacatttatg atactactat
gtgtatgtat 960 gaggtcatta aagccggtgg tttcacaaat ggcggcctga
actttgatgc gaaagctcgt 1020 aggggttcat tcacgcctga agatattttc
tatagttaca ttgctggtat ggatgctttc 1080 gcgttagggt ttagagcagc
tcttaaattg attggagacg gtagaattga caagtttgtg 1140 gcggataggt
atgcatcttg gaataccggt attggtgcag atattattgc cggaaaagcc 1200
gattttgcat cattggaaaa atatgctttg gaaaaaggtg aagttaccgc gtcattgtct
1260 tcaggtaggc aagagatgct ggaatctatt gtcaacaacg tattgtttag tttgtaa
1317 <210> SEQ ID NO 19 <211> LENGTH: 438 <212>
TYPE: PRT <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic polypeptide
<400> SEQUENCE: 19 Met Glu Phe Phe Ser Asn Ile Gly Lys Ile
Gln Tyr Gln Gly Pro Lys 1 5 10 15 Ser Thr Asp Pro Leu Ser Phe Lys
Tyr Tyr Asn Pro Glu Glu Val Ile 20 25 30 Asn Gly Lys Thr Met Arg
Glu His Leu Lys Phe Ala Leu Ser Trp Trp 35 40 45 His Thr Met Gly
Gly Asp Gly Thr Asp Met Phe Gly Cys Gly Thr Thr 50 55 60 Asp Lys
Thr Trp Gly Gln Ser Asp Pro Ala Ala Arg Ala Lys Ala Lys 65 70 75 80
Val Asp Ala Ala Phe Glu Ile Met Asp Lys Leu Ser Ile Asp Tyr Tyr 85
90 95 Cys Phe His Asp Arg Asp Leu Ser Pro Glu Tyr Gly Ser Leu Lys
Ala 100 105 110 Thr Asn Asp Gln Leu Asp Ile Val Thr Asp Tyr Ile Lys
Glu Lys Gln 115 120 125 Gly Asp Lys Phe Lys Cys Leu Trp Gly Thr Ala
Lys Cys Phe Asp His 130 135 140 Pro Arg Phe Met His Gly Ala Gly Thr
Ser Pro Ser Ala Asp Val Phe 145 150 155 160 Ala Phe Ser Ala Ala Gln
Ile Lys Lys Ala Leu Glu Ser Thr Val Lys 165 170 175 Leu Gly Gly Asn
Gly Tyr Val Phe Trp Gly Gly Arg Glu Gly Tyr Glu 180 185 190 Thr Leu
Leu Asn Thr Asn Met Gly Leu Glu Leu Asp Asn Met Ala Arg 195 200 205
Leu Met Lys Met Ala Val Glu Tyr Gly Arg Ser Ile Gly Phe Lys Gly 210
215 220 Asp Phe Tyr Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys His Gln
Tyr 225 230 235 240 Asp Phe Asp Thr Ala Thr Val Leu Gly Phe Leu Arg
Lys Tyr Gly Leu 245 250 255 Asp Lys Asp Phe Lys Met Asn Ile Glu Ala
Asn His Ala Thr Leu Ala 260 265 270 Gln His Thr Phe Gln His Glu Leu
Arg Val Ala Arg Asp Asn Gly Val 275 280 285 Phe Gly Ser Ile Asp Ala
Asn Gln Gly Asp Val Leu Leu Gly Trp Asp 290 295 300 Thr Asp Gln Phe
Pro Thr Asn Ile Tyr Asp Thr Thr Met Cys Met Tyr 305 310 315 320 Glu
Val Ile Lys Ala Gly Gly Phe Thr Asn Gly Gly Leu Asn Phe Asp 325 330
335 Ala Lys Ala Arg Arg Gly Ser Phe Thr Pro Glu Asp Ile Phe Tyr Ser
340 345 350 Tyr Ile Ala Gly Met Asp Ala Phe Ala Leu Gly Phe Arg Ala
Ala Leu 355 360 365 Lys Leu Ile Gly Asp Gly Arg Ile Asp Lys Phe Val
Ala Asp Arg Tyr 370 375 380 Ala Ser Trp Asn Thr Gly Ile Gly Ala Asp
Ile Ile Ala Gly Lys Ala 385 390 395 400 Asp Phe Ala Ser Leu Glu Lys
Tyr Ala Leu Glu Lys Gly Glu Val Thr 405 410 415 Ala Ser Leu Ser Ser
Gly Arg Gln Glu Met Leu Glu Ser Ile Val Asn 420 425 430 Asn Val Leu
Phe Ser Leu 435
* * * * *
References